Anti-PD-1 Antibodies and Fusion Proteins

ABSTRACT

Provided herein are recombinant antibodies, antigen-binding fragments, and fusion proteins thereof useful for binding to and inhibiting programmed death 1 (PD-1), nucleic acid molecules encoding the same and therapeutic compositions thereof, as well as methods of using such antibodies, including the methods for enhancing T cell and NK cell function to increase cell and cytokine mediated immunity and methods of treatment of various immune dysfunction related disorders including cancer and infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase entry pursuant to 35 U.S.C. § 371of International Application No. PCT/US2021/038740, filed Jun. 23, 2021,which claims the benefit of priority to U.S. Provisional PatentApplication No. 63/043,114, filed Jun. 23, 2020, and to U.S. ProvisionalPatent Application No. 63/111,459, filed Nov. 9, 2020, which areincorporated by reference herein in their entireties.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in txt format. Said txt copy, created Jun. 28, 2023, is named“2023-06-28_01183-0208-00US-KAD_ST25_ST25” and is 435,942 bytes in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

The present disclosure relates generally to the field of molecularbiology and medicine. More particularly, the disclosure provides fusionproteins comprising an anti-PD-1 antibody or antigen-binding fragmentthereof linked to an IL-15 polypeptide, which in turn is linked to anIL-15 receptor alpha (IL-15Rα) polypeptide comprising the IL-15Rα sushidomain. In addition, the disclosure provides antibodies, andantigen-binding fragments thereof, that specifically bind to PD-1 aswell as fusion proteins comprising such anti-PD-1 antibodies and PD-1binding fragments thereof. Also disclosed are therapeutic compositionscomprising the antibody fusions or antibodies for treating disease.

BACKGROUND

PD-1 is a 55 KD type I transmembrane protein of 286 amino acids, whichcontains a membrane proximal immunoreceptor tyrosine inhibitory motif(ITIM) and a membrane distal tyrosine-based switch motif (ITSM). ThePD-1 cytosolic domain contains two tyrosines, with the closest tyrosine(VAYEEL in mouse PD-1) located within the ITIM. The human PD-1 proteinand the murine PD-1 protein share about 60% amino acid identity, withfour potential N-glycosylation sites and residues that define the Ig-Vdomain are conserved. ITIM-like motifs surrounding the ITIM andC-terminal tyrosine in the cytoplasmic region are also conserved betweenhuman orthologue and murine orthologue. PD-1 is a member of CD-28 familyof receptors, which is primarily expressed on mature T cells inperipheral tissues and the tumor microenvironment. PD-1 is alsoexpressed on other non-T cell subsets including B cells, professionalantigen presenting cells (APCs), and natural killer (NK) cells.

The ligands for PD-1 are the B7 family members PD-L1 (also known asB7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). PD-L1 isexpressed primarily on both lymphoid and non-lymphoid tissues such asCD4 and CD8 T cells, macrophage lineage cells, peripheral tissues aswell as on tumor cells, virally infected cells and autoimmune tissuecells. However, PD-L2 has a more restricted expression than PD-L1, beingexpressed only on macrophages and activated dendritic cells. PD-1ligands are expressed in many human cancers, including melanoma, glioma,non-small cell lung cancer, squamous cell carcinoma of head and neck,leukemia, pancreatic cancer, renal cell carcinoma, and hepatocellularcarcinoma, and may be inducible in nearly all cancer. Interactionbetween PD-1 and its ligands results in dephosphorylation andinactivation of the T cell kinase ZAP70, and the recruitment of SHP2.SHP2 directly dephosphorylates PI3K, which inhibits downstreamactivation of Aktin causing a decrease in tumor infiltratinglymphocytes, and a decrease in T cell receptor mediated proliferation,leading to immune evasion. Inhibiting interaction between PD-1 and PD-L1reverses immune suppression and the effect may be additive when theinteraction of PD-1 with PD-L2 is blocked as well.

Several commercial anti-PD-1 antibodies are currently used to treatdiseases that involve PD-1-mediated immune suppression. However, only asubset of patients responds well to these therapies. Depending on theindication and other factors, the typical response rate of patients foreach monotherapy ranges from 10% to 30%. One possible reason for thislack of response could be the absence of an inflammatory tumormicroenvironment (TME), including activated anti-tumor CD8⁺ and CD4⁺ Tcells as well as other effectors such as NK cells.

Combination therapies involving PD-1 antibodies with various cytokinessuch as IL-2, IL-15, IL-21, tumor necrosis factor (TNF), andgranulocyte-macrophage colony-stimulating factor (GM-CSF) may have someefficacy in treating cancer and infection. However, these therapies arelimited by the systemic toxicity that is associated both with the highblood concentrations of cytokines that is required to obtain efficacyand with the lack of specificity of the administered cytokine foraffected cells and tissues.

IL-15 is a 12.5 KD glycoprotein with 114 amino acids and belongs to thefour α-helix bundle family of cytokines that also includes IL-2, IL-4,IL-7, IL-9, granulocyte colony-stimulating factor (G-CSF), and GM-CSF.IL-15 is secreted by macrophages, dendritic cells, and monocytes. IL-15can stimulate central memory CD8 cells to exert immunity withoutmodulating effects on other T cells. Additionally, IL-15 can activate NKcells and effector and memory CD8 T cells and can rescue T cells fromapoptosis induced by regulatory T cells (Tregs). Administration of IL-15is also associated with a lower risk of inducing systemic toxicity at ahigher dose compared to other cytokines. Human IL-15 can be soluble ormembrane-bound. The membrane-bound I1-15, which is the major form ofIL-15, is either formed by binding of IL-15 to cellular membranedirectly or by presentation of IL-15 by the membrane-bound IL-15Rreceptor.

The I1-15 receptor is composed of three subunits: IL-15Rα, IL-15R, andIL-15Rγ. IL-15 typically forms a complex with IL-15 receptor α expressedon APCs prior to binding to functional IL-15Rβ and γ units on T cellsand NK cells. IL-15 can bind to IL-15Rα receptor alone with affinity(K_(a)=1.1011 M⁻¹). It can also bind to IL-15Rβγc signaling complex withlower affinity (K_(a)=1.109 M⁻¹). The sushi domain (29.5 KD) of theIL-15Rα plays a critical role in complex formation of IL-15 and IL-15Rα.

One of the limitations with systemic IL-15 treatment is its very shorthalf-life in vivo. Therefore, there is a need to generate a suitableimmune-stimulatory form of IL-15/IL-15Rα that has a longer half-life invivo while retaining its ability to medulate the immune response.Additionally, there is a need for effective IL-15 antagonists that canbe selectively targeted to the disease site to avoid unwanted systemictoxicities and provide a more effective therapeutic benefit.

Compounds that modulate PD-1 activity have potential as therapeuticagents for the treatment of various diseases and disorders, includingcancer, inflammation, and autoimmune diseases. There is a significantunmet need to develop new strategies to target various effectormolecules to a disease site to provide therapeutic benefit without theside effects associated with non-specific immune activity.

SUMMARY

Provided herein are fusion proteins, antibodies, and antigen-bindingfragments thereof that bind to PD-1. In embodiments, the fusion proteinscomprise an anti-PD-1 antibody, or antigen-binding fragment thereof.Also disclosed are anti-PD-1 antibodies and the antigen-bindingfragments thereof that bind PD-1. Also provided are therapeuticcompositions of such fusion proteins, antibodies, and antigen-bindingfragments thereof, as well as methods of using these fusion proteins,antibodies and antigen-binding fragments thereof.

In one aspect, the disclosure provides a fusion protein comprising aPD-1 binding protein, an IL-15 receptor (IL-15R) binding protein (e.g.,IL-15 or an IL-15R binding portion thereof), and an IL-15 bindingprotein (e.g., IL-15Rα or an IL-15 binding portion thereof comprisingthe IL-15Rα sushi domain). In one aspect, the PD-1 binding protein is anantibody or PD-1 binding fragment thereof. In one embodiment, the IL-15receptor-binding portion is IL-15. In embodiments, IL-15 is joined,directly or indirectly, to the sushi domain of IL-15Rα.

In one embodiment, the fusion protein comprises an anti-PD-1 antibodyand an IL-15 polypeptide, wherein the C-terminus of the IL-15polypeptide is covalently linked, directly or via a polypeptide linker,to the N-terminus of the heavy chain variable region of one of theantibody heavy chains (i.e., the antibody heavy chains form aheterodimer wherein one of the heavy chains is linked to an IL-15polypeptide. In one embodiment, the N-terminus of the IL-15 polypeptideis linked to the C-terminus of an IL-15Rα sushi domain polypeptide.

In one embodiment, the fusion protein comprises an anti PD-1 antibodyand two IL-15 polypeptides, wherein each IL-15 polypeptide is covalentlylinked to the N-terminus of an antibody heavy chain.

In embodiments in which only one heavy chain is linked to IL-15/IL-15Rαsushi domain, the Fc domains of the antibody may comprise one or moreamino acid substitutions promoting heterodimer formation (i.e., theassociation of a heavy chain fusion with a heavy chain lacking thefusion). In embodiments, an amino acid residue in the CH3 domain of oneheavy chain (CH3-1) comprises an amino acid substitution replacing anamino acid with an amino acid residue having a larger side chain volume,thereby generating a “knob” within the CH3 domain, which is positionablein a “hole” present within the CH3 domain of the other heavy chain(CH3-2), generated by replacing an amino acid residue with an amino acidresidue having a smaller side chain volume. In embodiments, the aminoacid substitutions are selected from the following groups:

-   -   (1) CH3 domain of first heavy chain: S354C, T366W; CH3 domain of        second heavy chain: Y349C, T366S, L368A, Y407V    -   (2) CH3 domain of first heavy chain: T350V, L351Y, F405A, Y407V;        CH3 domain of second heavy chain: T350V, T366L, K392L, T394W    -   (3) CH3 domain of first heavy chain: L351Y, F405A, Y407V; CH3        domain of second heavy chain: T366L, K392L, T394W.

Either the first or second heavy chain may be linked to theIL-15/IL-15Rα sushi domain.

In embodiments, the IL-15Rα polypeptide and the IL-15 polypeptide arejoined by a first linker. In embodiments, the IL-15 polypeptide and theanti-PD-1 antibody, or antigen-binding portion thereof, are joined by asecond linker. In embodiments, the length of the linker mayindependently be between 10 to 40 amino acids. In embodiments, thelength of the linker is 25 to 35 amino acids. In embodiments, the linkersequences comprises near neutral amino acids selected from the groupcomprising Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln(Q), most preferably in the group comprising Gly (G), Asn (N), and Ser(S). In certain embodiments, the linker sequences are glycine and serinerich, and in some embodiments, the linker contains only serine andglycine residues.

In certain embodiments, the IL-15 and/or the IL-15Rα sushi domaincomprise one or more amino acid substitutions. In embodiments, the aminoacid substitutions are located at position 1, 4, 8, 30, 45, 61, 64, 65,and/or 108 of IL-15. In some embodiments, the amino acid substitutionsare located at positions 45, 65, and/or 108 of IL-15. In someembodiments the amino acid substitutions are one or more of N1D, N4D,D8N, D30N, D61N, E64Q, L45A, N65S/A/D/K, and/or Q108S/E substitutions inIL-15. In some embodiments, the amino acid substitutions are L45A,N65S/A/D/K, and/or Q108S substitutions in IL-15. In one embodiment, theamino acid substitution is an N65S substitution in IL-15.

In one embodiment, the amino acid substitution is an N60 substitution inthe IL-15Rα sushi domain. In one embodiment, the amino acid substitutionis an N60A substitution in the IL-15Rα sushi domain.

In embodiments, the fusion protein comprises an IL-15 polypeptidecomprising one of the following amino acid substitutions: N65S, N65A, orN65D. In embodiments, the fusion protein comprises an IL-15 polypeptidecomprising one or more of the following amino acid substitutions: (1)L45A, (2) N65S, N65A, or N65D, and (3) Q108S. In one embodiment, thefusion protein comprises an IL-15Rα sushi domain polypeptide comprisingsan N60A amino acid substitution.

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;    -   (ii) an IL-15 polypeptide or derivative thereof;    -   (iii) an IL-15Rα sushi domain polypeptide or derivative thereof;        and    -   (iv) a linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide or derivative thereof.

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214; and    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide.

The anti-PD-1 antibody, or antigen-binding fragment thereof, portion ofthe fusion protein can be any antibody, or antigen-binding fragmentthereof, that specifically binds PD-1, including those comprising thecorresponding heavy and light chain variable regions and/or CDRsprovided in FIG. 1 , or otherwise described herein. In embodiments, thefusion protein comprises an anti-PD-1 antibody, or antigen-bindingfragment thereof, wherein the CDRs of the heavy and light chain variableregions comprise the following sequences:

-   -   the sequence of CDR1 of the heavy chain variable region (CDR1H)        comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2 of the heavy chain variable region (CDR2H)        comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR2 of the heavy chain variable region (CDR3H)        comprises the sequence of SEQ ID NO: 23;    -   the sequence of CDR1 of the light chain variable region (CDR1L)        comprises the sequence RX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₈WX₁₉X₂₀ (SEQ ID        NO:135);        -   wherein X₁₃ is A or V;        -   wherein X₁₄ is S or G;        -   wherein X₁₅ is Q, E, or R;        -   wherein X₁₆ is G, S, D or N;        -   wherein X₁₇ is G, S, or N;        -   wherein X₁₈ is S, I, R, T, K, P, N, H, or V;        -   wherein X₁₉ is L or V; and        -   wherein X₂₀ is G or A; and    -   the sequence of CDR2 of the light chain variable region (CDR2L)        comprises the sequence X₂₁AX₂₂X₂₃X₂₄X₂₃X₂₆ (SEQ ID NO: 136); and        -   wherein X₂₁ is S, D, E, or A;        -   wherein X₂₂ is S or K;        -   wherein X₂₃ is S, N, T, R, or D;        -   wherein X₂₄ is L or V;        -   wherein X₂₅ is Q, E, or H; and        -   wherein X₂₆ is S, N, A, R, P, or T; and    -   the sequence of CDR3 of the light chain variable region (CDR3L)        comprises the sequence QQX₂₇X₂₈SFPX₂₉X₃₀ (SEQ ID NO: 137);        -   wherein X₂₇ is A or G;        -   wherein X₂₈ is N, D, or Y;        -   wherein X₂₉ is F or L; and        -   wherein X₃₀ is A or T.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy andlight chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   the light chain variable region comprises SEQ ID NO: 98, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        98.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequences of CDR1H, CDR2H and CDR3H comprise respectively        SEQ ID NOS: 21, 22, and 23; and    -   the sequences of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 45, 46, and 47; SEQ ID NOS: 66, 67, and 68; SEQ ID        NOS: 70, 67, and 71; SEQ ID NOS: 73, 74, and 75; SEQ ID NOS: 77,        78, and 47; SEQ ID NOS: 80, 81, and 82; SEQ ID NOS: 77, 78, and        84; SEQ ID NOS: 77, 86, and 47; SEQ ID NOS: 88, 89, and 47; SEQ        ID NOS: 66, 67, and 47; SEQ ID NOS: 80, 92, and 75; SEQ ID NOS:        80, 94, and 71; SEQ ID NOS: 99, 100, and 47; SEQ ID NOS: 102,        103, and 104; SEQ ID NOS: 106, 103, and 47; SEQ ID NOS: 108,        103, and 47; SEQ ID NOS: 110, 111, and 75; SEQ ID NOS: 77, 103,        and 113; SEQ ID NOS: 77, 111, and 47; SEQ ID NOS: 116, 67, and        47; SEQ ID NOS: 118, 119, and 47; SEQ ID NOS: 80, 78, and 47;        SEQ ID NOS: 122, 103 and 47; SEQ ID NOS: 124, 125, and 75; SEQ        ID NOS: 127, 38, and 68; SEQ ID NOS: 129, 130, and 47; or SEQ ID        NOS: 132, 133, and 75.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   the light chain variable region comprises SEQ ID NO: 48, 69, 72,        76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105, 107, 109, 112,        114, 115, 117, 120, 121, 123, 126, 128, 131, or 134, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to any of SEQ        ID NO: 48, 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 101, 105,        107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131, or        134.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy andlight chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence        RSSX₁SLLX₂SNGX₃X₄YLD (SEQ ID NO:62),        -   wherein X₁ is Q or E;        -   wherein X₂ is H or Y;        -   wherein X₃ is Y or N; and        -   wherein X₄ is T or N;    -   the sequence of CDR2L comprises the sequence X₅X₆SX₇X₈X₉X₁₀ (SEQ        ID NO: 63),        -   wherein X₅ is L, Q or E;        -   wherein X₆ is S, A, or V;        -   wherein X₇ is H, N, T, or S;        -   wherein X₈ is R or L;        -   wherein X₉ is G, A, or H; and        -   wherein X₁₀ is S or T; and    -   the sequence of CDR3L comprises the sequence MQGX₁₁X₁₂WPYT (SEQ        ID NO: 64),        -   wherein X₁₁ is A, T, or S; and        -   wherein X₁₂ is H or R.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprise the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 41;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 49;        and    -   the sequence of CDR3 of the light chain variable region (CDR3L)        comprises the sequence of SEQ ID NO: 50.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   the light chain variable region comprises SEQ ID NO: 51, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        51.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17,        18, and 19, respectively; and    -   the sequence of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 41, 42, and 43; SEQ ID NOS: 41, 52, and 53; SEQ ID        NOS: 41, 55, and 56; or SEQ ID NOS: 58, 59, and 60.

In further embodiments the fusion protein comprises an anti-PD-1antibody, or antigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   the light chain variable region comprises SEQ ID NO: 44, 54, 57,        or 61, or a sequence that is at least 90%, at least 95%, at        least 96%, at least 97%, at least 98%, or at least 99% identical        to any one of SEQ ID NO: 44, 54, 57, or 61.

In another aspect the disclosure provides an anti-PD-1 antibody, or anantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment thereof binds to PD-1, and wherein the antibodyor antigen-binding fragment thereof comprises a heavy chain variableregion and a light chain variable region wherein each of the heavy chainand the light chain variable regions comprise a CDR1, CDR2, and CDR3 andwherein the antibody heavy chains comprise a constant region comprisingthree constant domains CH1, CH2, and CH3.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23;    -   the sequence of CDR1L comprises the sequence        RX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₈WX₁₉X₂₀ (SEQ ID NO:135);        -   wherein X₁₃ is A or V;        -   wherein X₁₄ is S or G;        -   wherein X₁₅ is Q, E, or R;        -   wherein X₁₆ is G, S, D or N;        -   wherein X₁₇ is G, S, or N;        -   wherein X₁₈ is S, I, R, T, K, P, N, H, or V;        -   wherein X₁₉ is L or V;        -   wherein X₂₀ is G or A; and    -   the sequence of CDR2L comprises the sequence X₂₁AX₂₂X₂₃X₂₄X₂₃X₂₆        (SEQ ID NO: 136); and        -   wherein X₂₁ is S, D, E, or A;        -   wherein X₂₂ is S or K;        -   wherein X₂₃ is S, N, T, R, or D;        -   wherein X₂₄ is L or V;        -   wherein X₂₅ is Q, E, or H; and        -   wherein X₂₆ is S, N, A, R, P, or T; and    -   the sequence of CDR3L comprises the sequence QQX₂₇X₂₈SFPX₂₉X₃₀        (SEQ ID NO: 137);        -   wherein X₂₇ is A or G;        -   wherein X₂₈ is N, D, or Y;        -   wherein X₂₉ is F or L; and        -   wherein X₃₀ is A or T.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   a light chain variable region comprising SEQ ID NO: 98, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        98.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequences of CDR1H, CDR2H and CDR3H comprise respectively        SEQ ID NOS: 21, 22, and 23; and    -   the sequences of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 45, 46, and 47; SEQ ID NOS: 66, 67, and 68; SEQ ID        NOS: 70, 67, and 71; SEQ ID NOS: 73, 74, and 75; SEQ ID NOS: 77,        78, and 47; SEQ ID NOS: 80, 81, and 82; SEQ ID NOS: 77, 78, and        84; SEQ ID NOS: 77, 86, and 47; SEQ ID NOS: 88, 89, and 47; SEQ        ID NOS: 66, 67, and 47; SEQ ID NOS: 80, 92, and 75; SEQ ID NOS:        80, 94, and 71; SEQ ID NOS: 99, 100, and 47; SEQ ID NOS: 102,        103, and 104; SEQ ID NOS: 106, 103, and 47; SEQ ID NOS: 108,        103, and 47; SEQ ID NOS: 110, 111, and 75; SEQ ID NOS: 77, 103,        and 113; SEQ ID NOS: 77, 111, and 47; SEQ ID NOS: 116, 67, and        47; SEQ ID NOS: 118, 119, and 47; SEQ ID NOS: 80, 78, and 47;        SEQ ID NOS: 122, 103 and 47; SEQ ID NOS: 124, 125, and 75; SEQ        ID NOS: 127, 38, and 68; SEQ ID NOS: 129, 130, and 47; or SEQ ID        NOS: 132, 133, and 75.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   a light chain variable region comprising SEQ ID NO: 48, 69, 72,        76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115,        117, 120, 121, 123, 126, 128, 131, or 134, or a sequence that is        at least 90%, at least 95%, at least 96%, at least 97%, at least        98%, or at least 99% identical to any one of SEQ ID NO: 48, 69,        72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114,        115, 117, 120, 121, 123, 126, 128, 131, or 134.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence        RSSX₁SLLX₂SNGX₃X₄YLD (SEQ ID NO:62),        -   wherein X₁ is Q or E;        -   wherein X₂ is H or Y;        -   wherein X₃ is Y or N; and        -   wherein X₄ is T or N;    -   the sequence of CDR2L comprises the sequence X₅X₆SX₇X₈X₉X₁₀ (SEQ        ID NO: 63),        -   wherein X₅ is L, Q or E;        -   wherein X₆ is S, A, or V;        -   wherein X₇ is H, N, T, or S;        -   wherein X₈ is R or L;        -   wherein X₉ is G, A, or H; and        -   wherein X₁₀ is S or T; and    -   the sequence of CDR3L comprises the sequence MQGX₁₁X₁₂WPYT (SEQ        ID NO: 64),        -   wherein X₁₁ is A, T, or S; and        -   wherein X₁₂ is H or R.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 41;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 49;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 50.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   a light chain variable region comprising SEQ ID NO: 51, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        51.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17,        18, and 19, respectively; and    -   the sequence of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 41, 42, and 43; SEQ ID NOS: 41, 52, and 53; SEQ ID        NOS: 41, 55, and 56; or SEQ ID NOS: 58, 59, and 60.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   a light chain variable region comprising SEQ ID NO: 44, 54, 57,        or 61, or a sequence that is at least 90%, at least 95%, at        least 96%, at least 97%, at least 98%, or at least 99% identical        to any one of SEQ ID NO: 44, 54, 57, or 61.

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprises the sequence of SEQ ID NO:            21;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            22;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            23;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            96;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            97; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions S354C and T366W (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprises the sequence of SEQ ID NO:            21;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            22;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            23;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            96;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            97; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions T350V, L351Y, F405A, and Y407V (Kabat EU            index numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T350V, T366L, K392L, and T394W (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprises the sequence of SEQ ID NO:            21;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            22;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            23;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            96;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            97; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions L351Y, F405A, and Y407V (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T366L, K392L, and T394W (Kabat EU index            numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:    -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23;    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions S354C and T366W (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprises the sequence of SEQ ID NO:            21;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            22;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            23;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            96;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            97; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions T350V, L351Y, F405A, and Y407V (Kabat EU            index numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T350V, T366L, K392L, and T394W (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprises the sequence of SEQ ID NO:            21;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            22;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            23;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            96;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            97; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            82;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions L351Y, F405A, and Y407V (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T366L, K392L, and T394W (Kabat EU index            numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions S354C and T366W (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions T350V, L351Y, F405A, and Y407V (Kabat EU            index numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T350V, T366L, K392L, and T394W (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, or an amino acid sequence that is at least 90%        or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 95% identical to SEQ ID NO: 214;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions L351Y, F405A, and Y407V (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T366L, K392L, and T394W (Kabat EU index            numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions S354C and T366W (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions T350V, L351Y, F405A, and Y407V (Kabat EU            index numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T350V, T366L, K392L, and T394W (Kabat EU            index numbering).

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the antibody or antigen-binding fragment        thereof comprises a heavy chain variable region and a light        chain variable region, wherein each of the heavy chain and the        light chain variable regions comprise a CDR1, CDR2, and CDR3;        and wherein:        -   the sequence of CDR1H comprise the sequence of SEQ ID NO:            17;        -   the sequence of CDR2H comprises the sequence of SEQ ID NO:            18;        -   the sequence of CDR3H comprises the sequence of SEQ ID NO:            19;        -   the sequence of CDR1L comprises the sequence of SEQ ID NO:            41;        -   the sequence of CDR2L comprises the sequence of SEQ ID NO:            49; and        -   the sequence of CDR3L comprises the sequence of SEQ ID NO:            50;    -   (ii) an IL-15 polypeptide comprising the amino acid sequence of        SEQ ID NO: 212, optionally wherein amino acid N65 of SEQ ID NO:        212 is substituted with an S, D, or A;    -   (iii) an IL-15Rα sushi domain polypeptide comprising the amino        acid sequence of SEQ ID NO: 214, optionally wherein amino acid        N28 of SEQ ID NO: 214 is substituted with an A;    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide; and    -   optionally wherein the anti-PD-1 antibody, or an antigen-binding        fragment thereof, comprises two heavy chains, wherein:        -   (a) the CH3 domain of first heavy chain comprises the amino            acid substitutions L351Y, F405A, and Y407V (Kabat EU index            numbering); and        -   (b) the CH3 domain of second heavy chain comprises the amino            acid substitutions T366L, K392L, and T394W (Kabat EU index            numbering).

In one embodiment, the anti-PD-1 antibody or antigen-binding fragmentthereof provided by the disclosure is a multispecific or a bispecificantibody or antigen-binding fragment thereof. In one embodiment, theantibody or antigen-binding fragment is a bispecific antibody comprisinga complementary region that binds to PD-L1 or PD-L2. The disclosure alsoprovides conjugates of the antibodies or the fusion molecules, forexample, and without limitation, to fluorescent labels, imaging agents,therapeutic agents, or cytotoxic agents.

The disclosure further provides pharmaceutical compositions comprisingone or more of an anti-PD-1 antibody or antigen-binding fragmentthereof, or fusion protein comprising an anti-PD-1 antibody orantigen-binding fragment thereof, and a pharmaceutically acceptablecarrier.

In embodiments, the disclosure provides nucleic acid molecules encodingthe anti-PD-1 antibodies or antigen-binding fragments thereof disclosedherein and nucleic acid molecules encoding the fusion proteins disclosedherein, as well as vectors comprising such nucleic acid molecules. Alsoprovided are cells comprising a vector encoding the anti-PD-1 antibodiesor antigen-binding fragments thereof disclosed herein, or the fusionproteins disclosed herein.

In embodiments, the disclosure provides a method of inhibiting bindingof PD-1 to a ligand of PD-1, the method comprising administering to asubject in need thereof an effective amount of an anti-PD-1 antibody, orantigen-binding fragment thereof, or a fusion protein disclosed herein.

In embodiments, the disclosure provides a method of treating a diseasein a subject in need thereof, the method comprising administering tosaid subject a therapeutically effective amount of a compositioncomprising an anti-PD-1 antibody, or antigen-binding fragment thereof,or a fusion protein disclosed herein in a pharmaceutically acceptableform.

In embodiments, the disclosure provides a method for increasing T cellactivation in a subject in need thereof, the method comprisingadministering an effective amount of an anti-PD-1 antibody, orantigen-binding fragment thereof, or a fusion protein disclosed herein.In one embodiment, the disclosure provides a method of stimulating theimmune system in a subject in need thereof, the method comprisingadministering to said subject an effective amount of a compositioncomprising an anti-PD-1 antibody, or antigen-binding fragment thereof,or a fusion protein disclosed herein in a pharmaceutically acceptableform. In one embodiment, the disclosure provides a method of reducingreplication of a virus in a subject in need thereof, the methodcomprising administering to the subject an effective amount of acomposition comprising an anti-PD-1 antibody, or antigen-bindingfragment thereof, or a fusion protein disclosed herein.

The disclosure also provides a method of inhibiting the interaction ofPD-1 with PD-L1 and/or PD-L2 in a subject, the method comprisingadministering to a subject in need thereof an effective amount of ananti-PD-1 antibody, or antigen-binding fragment thereof, or a fusionprotein disclosed herein. The disclosure further provides a method ofinhibiting immunosuppression mediated by PD-1 in a subject in needthereof, the method comprising administering to the subject an effectiveamount of an anti-PD-1 antibody, or antigen-binding fragment thereof, ora fusion protein disclosed herein.

The disclosure further provides a method of stimulating an immuneresponse against a cell or tissue that expresses PD-1 in a subject inneed thereof, the method comprising administering to the subject aneffective amount of an anti-PD-1 antibody, or antigen-binding fragmentthereof, or a fusion protein disclosed herein. In certain embodiments,the cell or tissue expressing PD-1 is a cancerous cell or a cellinfected with a pathogen, including, but not limited to, a virus or abacterium.

In one aspect, the present disclosure provides a method of treatingcancer, an immune disorder, or an infection in a patient in needthereof, the method comprising (a) treating a cell, in vitro, with ananti-PD-1 antibody, or antigen-binding fragment thereof, or a fusionprotein disclosed herein; and (b) administering the treated cell to thepatient. In some embodiments, the cell is a T cell.

In another aspect, the disclosure provides a method of increasing T cellactivation in response to an antigen in a subject, the method comprisingadministering to the subject an effective amount of an anti-PD-1antibody, or antigen-binding fragment thereof, or a fusion proteindisclosed herein, or a pharmaceutical composition disclosed herein.

In another aspect, the disclosure provides a method of treating acondition in a subject that would benefit from an upregulation of thesubject's immune response, the method comprising administering to thesubject an effective amount of an anti-PD-1 antibody, or antigen-bindingfragment thereof, or a fusion protein disclosed herein. In certainembodiments, the subject has upregulated expression of PD-L1, or thesubject has been identified as positive for expression of PD-L1.

In one embodiment, the disclosure provides methods of using theanti-PD-1 antibodies, or antigen-binding fragments thereof, or fusionproteins disclosed herein for treating a subject that does not respondto therapy with a checkpoint inhibitor (primary resistance), and/or fortreating a subject that initially responds to checkpoint inhibitortreatment, but later becomes resistant to checkpoint inhibitor blockade(secondary or acquired resistance). Such methods for treating compriseadministering to said subject an anti-PD-1 antibody, or antigen-bindingfragment thereof, or a fusion protein disclosed herein. In someembodiments, the subject has acquired resistance to therapy with one ormore of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

In one embodiment, the disclosure provides a method of administering ananti-PD-1 antibody, or antigen-binding fragment thereof, or a fusionprotein disclosed herein to a subject in need thereof, the methodfurther comprising administering an additional therapeutic agent ortherapy to the subject, wherein the additional therapeutic agent ortherapy is selected from the group consisting of a cancer vaccine, acheckpoint inhibitor, an antibody to a tumor-specific antigen, aBacillus Calmette-Guerin (BCG) vaccine, a cytotoxin, an interleukin 6receptor (IL-6R) inhibitor, an interleukin 4 receptor (IL-4R) inhibitor,an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, an antibody-drugconjugate, an anti-inflammatory drug, and a dietary supplement. Inembodiments, the disclosure provides a method of administering ananti-PD-1 antibody, or antigen-binding fragment thereof, or a fusionprotein disclosed herein to a subject in need thereof, the methodfurther comprising administering to the subject a checkpoint inhibitorselected from a CTLA-4, a PD-1, a PD-L1, and a PD-L2 inhibitor. Inembodiments, the disclosure provides a method of administering ananti-PD-1 antibody, or antigen-binding fragment thereof, or a fusionprotein disclosed herein to a subject in need thereof, the methodfurther comprising administering to the subject one or more of aninhibitor of LAG3, TIGIT, LAP, Podoplanin, Protein C receptor, ICOS,GITR, CD226, and/or CD160. In embodiments, the disclosure provides amethod of administering an anti-PD-1 antibody, or antigen-bindingfragment thereof, or a fusion protein disclosed herein to a subject inneed thereof, the method further comprising administering an additionaltherapeutic agent or therapy, wherein the additional therapeutic agentor therapy is administered concurrently or consecutively with theanti-PD-1 antibody, or antigen-binding fragment thereof, or fusionprotein disclosed herein. In embodiments, the disclosure provides amethod of administering an anti-PD-1 antibody, or antigen-bindingfragment thereof, or a fusion protein disclosed herein to a subject inneed thereof, the method further comprising administering to the subjectan additional therapeutic agent or therapy, wherein the additionaltherapeutic agent or therapy is administered separately, or as a mixturewith the anti-PD-1 antibody, or antigen-binding fragment thereof, orfusion protein disclosed herein. In one embodiment, the disclosureprovides a method of administering an anti-PD-1 antibody, orantigen-binding fragment thereof, or a fusion protein disclosed hereinin combination with chemotherapy, radiotherapy, or surgery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D show the heavy and light chainvariable domain sequences and CDR sequences of anti-PD-1 antibodiesdisclosed herein. CDR sequences are according to Kabat (CDR sequencesaccording to AbM/Chothia exclude the underlined amino acids). FIG. 1Aprovides the heavy and light chain variable regions of six anti-humanPD-1 (hPD-1) antibodies that were identified by library screening. FIG.1B provides the light chain and CDR sequences of 23H9 as well as of 23H9variants that were identified by light chain shuffling, wherein alibrary of light chains was paired with the 23H9 heavy chain sequence.X₁ is Q or E; X₂ is H or Y; X₃ is Y or N; X₄ is T or N; X₅ is L, Q or E;X₆ is S, A, or V; X₇ is H, N, T, or S; X₈ is R or L; X₉ is G, A, or H;X₁₀ is S or T; X₁₁ is A, T, or S; and X₁₂ is H or R. FIG. 1C providesthe light chain and CDR sequences of 24H9 as well as of 24H9 variantsthat were identified by light chain shuffling, wherein a library oflight chains was paired with the 24H9 heavy chain sequence. X₁₃ is A orV; X₁₄ is S or G; X₁₅ is Q, E, or R; X₁₆ is G, S, D or N; X₁₇ is G, S,or N; X₁₈ is S, I, R, T, K, P, N, H, or V; X₁₉ is L or V; X₂₀ is G or A;X₂₁ is S, D, E, or A; X₂₂ is S or K; X₂₃ is S, N, T, R, or D; X₂₄ is Lor V; X₂₅ is Q, E, or H; X₂₆ is S, N, A, R, P, or T; X₂₇ is A or G; X₂₈is N, D, or Y; X₂₉ is F or L; and X₃₀ is A or T. FIG. 1D providessequences for hPD-1/mPD-1 cross-reacting antibody R3A9 as well as R3A9derivatives that were identified by light chain shuffling, wherein alibrary of light chains was paired with the R3A9 heavy chain sequence.

FIG. 2A and FIG. 2B illustrate the binding of antibodies 19B6, 19B10,23A8, 23H9, 24H9, and 23A11 to hPD-1-Fc (FIG. 2A, labels as in FIG. 2B)as determined by ELISA, and to hPD-1-his (FIG. 2B).

FIG. 3 depicts the ability of antibodies 19B6, 19B10, 23A8, 23H9, 24H9,and 23A11 to block interaction between hPD-1 to hPD-L1.

FIG. 4A, FIG. 4B, and FIG. 4C illustrate the binding of antibodies 19B6,19B10, 23A8, 23H9, 24H9, and 23A11 to hPD-1-293 cells (FIG. 4A), toSEB-activated PBMC from donor “EA” (low PD-1 expression) (FIG. 4B), andto SEB-activated PBMC from donor “AF” (high PD-1 expression) (FIG. 4C),respectively. FIG. 4A: EC₅₀ 23A8>23H9>19B10. MFI 23A8>23H9>19B10. FIG.4B: 23H9>24H9 and 23A8. FIG. 4C: 23H9>24H9 and 23A8.

FIG. 5A and FIG. 5B illustrate the ability of antibodies 23A8 and 23H9(FIG. 5A) as well as 19B6 and 24H9 (FIG. 5B) to block interactionsbetween hPD-1 and hPD-L1.

FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D depict binding of antibodies23H9, 31B1, 33C4, 33G8, and 34C1 to hPD-1-Fc (FIG. 6A, same labels asFIG. 6B), binding to strepavidin-captured biotin-hPD-1-his (FIG. 6B),binding to ahis-captured hPD-1-his (FIG. 6C, same labels as FIG. 6D),and blocking of hPD-1-Fc/hPD-L1-Fc interaction (FIG. 6D). No significantdifference was observed between the parental antibody 23H9 and itsderivatives (FIG. 6A and FIG. 6B). All derivatives had lower EC₅₀ andhigher inhibition values than the parental antibody (FIG. 6C and FIG.6D).

FIG. 7 depicts binding of antibodies 24H9, 32A11, 32D2, 32D11, 38A6,38A10, 38A11, 38B2, 38C11, 39B3, 39B11, 39G8 and 38H11 to hPD-1-Fc(first, far left column), binding to strepavidin-captured hPD-1-his(second column), binding to ahis captured hPD-1-his (third column), andblocking of hPD-1/hPD-L1 interactions (fourth column).

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D depict binding of antibodies31B1, 33C4, 23H9, 38A6, 24H9, and 38B2 to hPD-1-293 cells (FIG. 8A, samelabels as FIG. 8B) and hPD-1 Jurkat cells (FIG. 8B) as well as bindingto SEB-activated PBMC from donor FA (FIG. 8C, same labels as FIG. 8D)and donor FB (FIG. 8D). FIG. 8A. MFI to hPD1-HEK293: 24H9 derivatives(38B2 and 38A6>24H9)>23H9 derivative (31B1 and 33C4>23H9). FIG. 8B. MFIto hP01-Jurkat: 23H9 derivatives (33C4>31B1>23H9)>24H9 derivatives(38B2>38A6>24H9). FIG. 8C and FIG. 8D. EC₅₀ value for binding toSEB-activated human PBMCs from two donors: 23H9 derivatives (31B1 sameas 33C4)<24H9 derivatives (38A6 same as 31B1). MFI for binding toSEB-activated human PBMCs: 23H9 derivatives (31B1 same as 33C4)>24H9derivatives (38A6 same as 31B1).

FIG. 9A and FIG. 9B illustrate the ability of antibodies 23H9, 31B1,33C4, 24H9, 38B2, and 38A6 to block PD-1/PD-L1 interactions using aPromega binding assay. FIG. 9A. EC₅₀ of 23H9 and its derivatives 31B1and 33C4: 31B1 (0.139 μg/ml)>33C4 (0.165 μg/ml)>23H9 (0.511 μg/ml).Maximum fold induction (luminescence): 31B1 (6.48)>33C4 (6.24)>23H9(5.56). FIG. 9B. EC₅₀ of 24H9 and its derivatives 38A6 and 38B2: 38B2(0.855 μg/ml)>38A6 (0.164 μg/ml)>24H9 (0.855 μg/ml). Maximum foldinduction (luminescence): 38B2 (6.54)>38A6 (5.66)>24H9 (4.97).

FIG. 10A, FIG. 10B, and FIG. 10C depict IL-2 accumulation for PBMCs fromdonor FA (FIG. 10A) and IL-2 accumulation for PBMCs from donor EA (FIG.10B and FIG. 10C), in presence of antibodies 23H9, 31B1, 24H9, and 38B2.FIG. 10A. IL-2 accumulation in PBMCs from donor FA: IL-2 concentrationincreased in the presence of the indicated antibodies in adose-dependent manner. FIG. 10B. 23H9 and 31B1: EC₅₀ was 0.344 and 0.107nM, respectively; IL-2 concentration at 10 nM antibody concentration was3.9 and 5.81 μg/ml, respectively. FIG. 10C. 24H9 and 38B2: EC₅₀ was >10nM and 0.019 nM, respectively; IL-2 concentration at in 10 nM antibodyconcentration was 4.5 and 5.0 μg/ml, respectively.

FIG. 11 illustrates the thermo-stability measurement for antibodies780_38 (31B1), 780_39 (33C4), 780_46 (38A6), and 780_54 (38B2) asdetermined by differential scanning calorimeter (DSC).

FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, and FIG. 12E depict binding ofantibodies 31B1, 33C4, 38B2, and 38A6 to hPD-1FC (FIG. 12A),streptavidin-captured biotin-labeled hPD-1his (FIG. 12B, labels as inFIG. 12A), and anti-his antibody captured hPD-1his (FIG. 12C, labels asin FIG. 12A) as well as blocking of hPD-1-hPD-L1 (FIG. 12D) andhPD-1-hPD-L2 interactions (FIG. 12E).

FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D depict dose-response bindingof antibodies 31B1, 33C4, 38A6, and 38B2 to hPD-1-293 cells (FIG. 13A),hPD-1-Jurkat cells (FIG. 13B), SEB-activated PBMCs from donor FA (FIG.13C), and SEB-activated PBMCs from donor FB (FIG. 13D). FIG. 13A. Allantibody derivatives shown have similar EC₅₀ values, but 24H9derivatives 38A6 and 38B2 exhibit higher maximum binding signals. Samelabels as in FIG. 13B. FIG. 13B. 23H9 derivatives 31B1 and 33C4 havelower EC₅₀ values and higher maximum binding signals as compared to 24H9derivatives 38A6 and 38B2. FIG. 13C. Same labels as in FIG. 13D. FIG.13C and FIG. 13D. 23H9 derivatives 31B1 and 33C4 exhibit higher maximumbinding signals and lower EC₅₀ values as compared to 24H9 derivatives38A6 and 38B2 as determined using PBMCs from two different donors.

FIG. 14A and FIG. 14B depict K_(D) values obtained for binding ofantibodies 38B2, 38A6, 33C4, and 31B1 to hPD-1-his (FIG. 14A) andhPD-1-Fc (FIG. 14B) as determined by SPR.

FIG. 15A and FIG. 15B illustrate the ability of antibodies 31B1 and 38B2to block intercellular hPD-1-hPD-L1 interactions. The EC₅₀ valuesaveraged from two experiments for 31B1 and 38B2 were 0.168 and 0.154μg/ml, respectively. Fold induction of luminescence was also averagedfrom two experiments. No significant difference between 31B1 and 38B2was observed. Both 31B1 and 38B2 were superior to control B (Nivolumab,sold under the brand name Opdivo) and similar to control M(Pembrolizumab, sold under the brand name Keytruda). FIG. 15A.Experiment 1. FIG. 15B. Experiment 2.

FIG. 16A and FIG. 16B illustrate the effect of anti-PD-1 antibodies31Bland 38B2 on IL-2 cytokine production in SEB-activated PBMCs fromdonor EA (FIG. 16A) and donor FA (FIG. 16B). Cytokine IL-2 secretion wasexamined in SEB-activated human PBMC cells from donor EA and FA. TheEC₅₀ values for 38B2, 31B1, ctM and ctB were 0.101, 0.297, 0.210 and0.548 nM (PBMC from donor EA). The maximum IL-2 secretion by cellstreated by 38B2, 31B1, ctM or ctB was 8.05, 7.73, 7.21 and 6.79 ng/ml,respectively. 38B2 was more potent than 31B1 and two control antibodies;31B1 was more potent than control B and comparable to control M.

FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F depictthe dose dependent binding of antibodies m3A7 and R3A9 to solublemPD-1-Fc (FIG. 17A), soluble strepatavidin-captured mPD-1-Fc (FIG. 17B),soluble streptavidin-captured mPD-1-his (FIG. 17C), cells expressingmPD-1 (FIG. 17C) as well as blocking of mPD-1 to PD-L1 by antibodiesm3A7 and R3A9 as determined by ELISA (FIG. 17E) and blockage ofintercellular mPD-1/PD-L1 interactions as determined by a Promega assay(FIG. 17F).

FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, and FIG. 18E illustrate bindingof antibodies 38B2, m2C1, m2A1, m4D1, m3A7, and R3A9 to hPD-1 (FIG. 18A,labels as in FIG. 18B), mPD-1 (FIG. 18B), and streptavidin-capturedbiotin mPD-1 (FIG. 18C, labels as in FIG. 18B) as determined by ELISA aswell as blocking of mPD-1-mPD-L1 interactions by antibodies 38B2, m2C1,m2A1, m4D1, m3A7, and R3A9 (FIG. 18D, labels as in FIG. 18E), andblocking of hPD-1-hPD-L1 interactions (FIG. 18E) as determined byELISA).

FIG. 19A, FIG. 19B, FIG. 19C, and FIG. 19D depict binding of antibodiesm2A1, m2C1, m4D1, R3A9, m3A7 and 38B2 to cells expressing hPD-1 ormPD-1. Cells expressing human PD-1: hPD-293 (FIG. 19A, labels as in FIG.19D) and hPD-1 Jurkat (FIG. 19B, labels as in FIG. 19D). Cellsexpressing murine PD-1: FIG. 19C (labels as in FIG. 19D) and FIG. 19D.

FIG. 20A and FIG. 20B depict blocking of mPD-1/mPD-L1 interactions andblocking of hPD-1-hPD-L1 interactions by antibodies 38B2, m4D1, andm2C1. FIG. 20A. Blockade: m4D1 was as potent as m3A7 in blockingmPDI-mPDL 1 interactions. IC50 m4D1=0.5724 nM. IC50 m3A7=0.5615 nM. IC50m2C1=1.127 nM. FIG. 20B. m4D1 blocks hPD1/hPDL 1 interactions but wasnot as potent as 38B2. IC50 38B2=0.7248 nM. IC50 m4D1=2.125 nM. IC50m2C1=31.25 nM.

FIG. 21A and FIG. 21B depict schematic diagrams of exemplary PD-1/IL-15fusion proteins.

FIG. 22A, FIG. 22B, FIG. 22C, and FIG. 22D depict binding of fusionproteins 1N-m3A7/IL-15, 2N-m3A7/IL-15, 1C-m3A7/IL-15, and 2C-m3A7/IL-15to mPD-1 (FIG. 22A) and to cells expressing mPD-1 (FIG. 22B) as well asblocking of mPD-L1 to mPD-1 using a competition ELISA (FIG. 22C) or aPromega blocking assay (FIG. 22D).

FIG. 23A, FIG. 23B. and FIG. 23C depict the effect of fusion proteins1N-m3A7/IL-15, 2N-m3A7/IL-15, 1C-m3A7/IL-15, and 2C-3A7/IL-15 onproliferation of CTLL2 (FIG. 23A) and C57BL6 (FIG. 23B). FIG. 23C.CD8⁺/CD4⁺ T cell populations in total spleen cells. Fusion proteins1N-DP47/IL-15, 2N-DP47/IL-15, 1C-DP47/IL-15, and 2C-DP47/IL-15 were usedas controls.

FIG. 24 depicts the ability of anti-PD-L1/SD15 fusion proteins to reducetumor growth of mice bearing PD-1 expressing tumors in a PD-1/PD-L1resistant Lewis Lung Model (LL2).

FIG. 25A and FIG. 25B illustrate that, as compared to C-terminalfusions, N-terminal fusion proteins are particularly useful to act ontumor infiltrating lymphocytes (TILs) compared to peripheral T cells.This is due to the cis-presentation enabled by the N-terminal fusionconstructs.

FIG. 26A, FIG. 26B, and FIG. 26C illustrate blocking of PD-1/PD-L1interactions (FIG. 26A and FIG. 26C) and PD-1/PD-L2 interactions (FIG.26B) for hPD-1fusion proteins (FIG. 26A and FIG. 26B) and mutated hPD-1fusion proteins (comprising (38B2)/N65D/S/A-mutated IL-15) (FIG. 26C).

FIG. 27A, FIG. 27B and FIG. 27C illustrate stimulation of theproliferation of M07e cells by the indicated anti-PD-L1-SD15 fusionproteins.

FIG. 28 illustrates the impact of different ofanti-PD-L1/IL-15/linker/IL-15Rα sushi domain fusion proteins on tumorsize. A CT26 syngeneic model was used. Single dose, IV. Anti-PD-L1antibody: D7A8.

FIG. 29A, FIG. 29B, FIG. 29C, FIG. 29D, FIGS. 29E, and 29F illustratethat the presence of an N65S mutation in IL-15 in a 1N-fusion proteindoes not affect the ability of the fusion to bind PD-1 and to disruptPD-1 interactions. FIG. 29A. Binding of 1N-fusion proteins to a solublehPD-1-Fc. FIG. 29B. ELISA competition assay for measuring PD-L1 bindingto PD-1. FIG. 29C. ELISA competition assay for measuring PD-L2 bindingto PD-1.

FIG. 29D. Binding of 1N-fusion proteins to hPD-1 transfected HEK293cells. FIG. 29E. Promega blockade assay to determine binding of PD-L2 toPD-1. FIG. 29F. Promega blockade assay to determine binding of PD-L2 toPD-1. Fusion proteins comprising antibody DP47 (a non-targeting controlantibody) served as controls. mut=N65S.

FIG. 30A, FIG. 30B, FIG. 30C, FIG. 30D, and FIG. 30E illustrate that thepresence of an N65S mutation in IL-15 in a 1N-fusion protein leads tosignificant potency decreases of the fusion protein in proliferatinghuman cells. FIGS. 30A, 30B, and 30C show stimulation of proliferationof M07e cells by anti-PD-1 (38B2)/N65D/S/A-mutated IL-15 fusions. Fusionproteins comprising a non-targeting control antibody served as controls(FIG. 30B). FIG. 30D shows proliferation of hPBMCs upon stimulation withthe indicated fusion proteins. FIG. 30E shows binding of the indicatedfusion proteins to hIL2Rβ transfected HEK30 cells.

FIG. 31A, FIG. 31B, FIG. 31C, and FIG. 31D illustrate that the presenceof an N65S mutation in IL-15 in a 1N-fusion protein leads to significantpotency decreases of the fusion protein in proliferating murine cellsand an increase in dose response in proliferating CD8⁺ T cells than CD4⁺T cells. FIGS. 31A and 31B show proliferation of murine CTLL2 cells(FIG. 31A) and murine BALB/c spleen cells (FIG. 31B) upon stimulationwith the indicated fusion proteins. FIGS. 31C and 31D show CD8⁺ and CD4⁺T cell populations in murine BALB/c spleen cells (FIG. 31C) and hPBMCs(FIG. 31D) upon stimulation with the indicated fusion proteins.

FIG. 32A, FIG. 32B, FIG. 32C, FIG. 32D, FIG. 32E, FIG. 32F, FIG. 32G,FIG. 32H, FIG. 32I, FIG. 32J, and FIG. 32K illustrate that 1N-fusionproteins comprising an IL-15 mutation exhibited strong anti-tumoractivity in vivo in an anti-PD-1 antibody-resistant mouse model. FIGS.32A, FIG. 32B, FIG. 32F, FIG. 32G, FIG. 32I, and FIG. 32K. Tumor volumeafter treatment with Keytruda or the indicated 1N-fusion proteins.mut=N65S mutation. ct=DP47=non-targeting control antibody.Ketruda=pembrolizumab (formerly lambrolizumab, brand name Keytruda).FIG. 32C. Tumor growth for each mouse (N65S-38B2/IL-15, 12 mg/kgtreatment). FIG. 32D, FIG. 32H, and FIG. 32J. % change in body weight.FIG. 32E. Mouse survival at different dose levels. FIGS. 32A, FIG. 32B,FIG. 32C, FIG. 32D, FIG. 32E, FIG. 32F, FIG. 32G, FIG. 32H, FIG. 32I:Dosing was QW=once per week intraperitoneally for three weeks for1N-fusion proteins, BIW=twice per week intraperitoneally for three weeksfor controls. FIG. 32J, and FIG. 32K: Dosing was BIW=twice per weekintraperitoneally for three weeks.

FIG. 33 shows that N65S-1N-38B2/IL-15 and N65D-1N-38B2/IL-15 fusionproteins prevent a re-challenge in mice bearing a hPDL1-CT26 tumor.

FIG. 34A. FIG. 34B, and FIG. 34C show that 1N-fusion proteins comprisingan IL-15 mutation exhibited improved anti-tumor activity in hPD1/PDL1mice bearing hPDL1-CT26 tumor as compared to combinations ofnon-targeted fusion protein and an anti-PD-1 antibody. FIG. 34A. Tumorvolume after treatment with the indicated proteins. FIG. 34B. Mousesurvival after treatment with the indicated proteins. FIG. 34C. % changein body weight after treatment with the indicated proteins.

FIG. 35A. FIG. 35B, and FIG. 35C show that the additional administrationof an anti-PD-1 antibody does not lead to a further increase inanti-tumor activity of 1N-fusion proteins. FIG. 35A. Tumor volume aftertreatment with the indicated proteins. FIG. 35B. Mouse survival aftertreatment with the indicated proteins. FIG. 35C. % change in body weightafter treatment with the indicated proteins.

FIG. 36A and FIG. 36B illustrate that N65S-1N-38B2/IL-15 exhibited asimilar serum half-life profile as antibody 38B2 in both IV (FIG. 36A)and IP (FIG. 36B) injection with dose response up to 120 h aftertreatment.

FIG. 37A, FIG. 37B, FIG. 37C, and FIG. 37D illustrate the results of amechanism of action study in hPDL1/PD1 transgenic BALB/c mice bearinghPDL1-CT26 tumors. FIG. 37A. 0.5×10⁶ of hPDL1/CT26 cells weresubcutaneously inoculated to the right lower flank of the hPD1/PDL1transgenic BALB/c mice. When the tumor size reached about 175 mms, themice were intravenously injected with of (1) N65S-1N-38B2/IL-15 (0.06μmol/kg and 0.035 μmol/kg, respectively), (2) N65S-1N-DP47/IL-15(non-targeted control, 0.035 μmol/kg), (3) antibody 38B2 (0.035 μmol/kg)and (4) the combination of antibody 38B2 (0.035 μmol/kg) andN65S-1N-38B2/IL-15 (0.035 μmol/kg). FIG. 37B. Blood and draining lymphnode (DLN) from all groups were collected to perform flow cytometry.Tumors were collected to perform TILs analysis. FIG. 37C. CD8/CD4 ratioin tumors, the blood, and draining lymph nodes (treated with antibody(1) 38B2, (2) PD1 targeted fusion protein N65S-1N-38B2/IL-15, (3)non-targeted fusion protein N65S-1N-DP47/IL-15, or (4) a combination of38B2 and N65S-1N-38B2/IL-15). FIG. 37D. Analysis of CD8 and CD4 subtypesin tumors (treated with antibody (1) 38B2, (2) PD1 targeted fusionprotein N65S-1N-38B2/IL-15, (3) non-targeted fusion proteinN65S-1N-DP47/IL-15, or (4) a combination of 38B2 andN65S-1N-38B2/IL-15).

FIG. 38 illustrates that a N65S-1N-m3A7/IL-15 (surrogate) fusion proteinshowed significant anti-tumor efficacy in numerous mouse syngeneic tumormodels. Data for EMT6 as shown as an illustrative example.

DETAILED DESCRIPTION

The present disclosure provides fusion proteins comprising an anti-PD-1antibody or antigen-binding fragment thereof linked to an IL-15polypeptide, which in turn is linked to an IL-15 receptor alpha(IL-15Rα) polypeptide comprising the IL-15Rα sushi domain. In addition,provided herein are antibodies, and antigen-binding fragments thereof,that specifically bind to PD-1 and fusion proteins comprising suchanti-PD-1 antibodies and PD-1 binding fragments thereof.

Fusion Proteins Comprising Anti-PD-1 Antibodies or Antigen-FragmentsThereof

In one aspect, the disclosure provides fusion proteins comprising anantibody, or antigen-binding fragment thereof that binds to PD-1. Thisportion of the fusion protein can be any antibody or antibody fragmentthat specifically binds PD-1, including those comprising thecorresponding heavy and light chain variable regions or CDRs provided inFIG. 1 , or otherwise described herein.

Disclosed herein are fusion proteins comprising a stimulatory domain. Asused herein, a “stimulatory domain” is a domain that promotes an immuneresponse. The stimulatory domain may stimulate an immune responsemediated by, for example, inducing T cell or NK cell activity and/orproliferation. In embodiments, the stimulatory domain stimulates cellsthat respond to an interleukin or an interferon, such as, withoutlimitation, IL-2, IL-7, IL-15, and IL-21. In one embodiment, thestimulatory domain binds to and stimulates a receptor that is responsiveto an interleukin or an interferon, such as, without limitation, IL-2,IL-7, IL-13, IL-15, and IL-21. The stimulatory domain can also be ahybrid domain that is a hetero-complex of two or more ligands covalentlylinked to each other.

In embodiments, the stimulatory domain includes a sequence or domainthat promotes IL-15 stimulation of the IL-15 receptor (IL-15R).

In one embodiment, the stimulatory domain that promotes IL-15Rstimulation comprises IL-15 or an IL-15 derivative.

In one embodiment, the stimulatory domain that promotes IL-15Rstimulation comprises an IL-15Rα polypeptide comprising the IL-15Rαsushi domain or a derivative thereof. In an embodiment, the stimulatorydomain comprises the sushi domain of the IL-15Rα chain.

In an embodiment, the stimulatory domain comprises IL-15 or a derivativethereof, whose binding may be enhanced by the presence of an IL-15Rαpolypeptide comprising the IL-15Rα sushi domain or a derivative thereof.In one embodiment, the stimulatory domain comprises a complex of IL-15or a derivative thereof and an IL-15Rα polypeptide comprising theIL-15Rα sushi domain or a derivative thereof. In some embodiment, thestimulatory domain comprises an IL-15 or a derivative thereof and anIL-15Rα polypeptide comprising the IL-15Rα sushi domain or a derivativethereof, wherein the two polypeptides are covalently linked by a linker(including, but not limited to, a linker comprising SEQ ID NO: 215).

In one embodiment, the IL-15 or a derivative thereof is locatedN-terminally of the IL-15Rα polypeptide comprising the IL-15Rα sushidomain or derivative thereof. In one embodiment, the IL-15 or aderivative thereof is located C-terminally of the IL-15Rα polypeptidecomprising the IL-15Rα sushi domain or derivative thereof. In oneembodiment, the stimulatory domain comprises the sequence of the SD15domain highlighted in any one of SEQ ID NOS:177-180.

As demonstrated herein, provided herein is a fusion protein whichcomprises (1) a PD-1 binding domain that blocks binding of PD-1 to PD-L1and inhibits immunosuppression, and (2) a stimulatory domain thatpromotes an immune response, wherein the fusion proteins provides forincreased immune cell activity, compared to two distinct molecules thatprovide the before mentioned functions separately. Specifically, theexperiments disclosed herein demonstrate that fusion proteins containingboth a PD-1 binding domain that blocks binding of PD-1 to PD-L1, and anstimulatory domain IL-15 or a derivative thereof and an IL-15Rαpolypeptide comprising the IL-15Rα sushi domain or a derivative thereof,promoted increased proliferation, Th1 cytokine release, and killingactivity-related molecules of NK and T cells, compared to providing thedomains in separate molecules.

In certain embodiments, the fusion protein comprises a stimulatorydomain disclosed herein that is covalently linked to an anti-PD-1antibody or antigen-binding fragment thereof disclosed herein via aflexible linker. In some embodiments, provided herein is a fusionprotein wherein the stimulatory domain disclosed herein is fuseddirectly to an anti-PD-1 antibody or antigen-binding fragment disclosedherein.

As used herein, “covalently linked” or “fused” refers to the associationof two or more polypeptides through a covalent bond. In someembodiments, two polypeptides that are covalently linked are fused toeach other directly, i.e., without any additional polypeptide sequencebetween the first and the second peptide. Accordingly, in someembodiments, the N-terminus of the first polypeptide is fused directlyto the C-terminus of the second polypeptide or vice versa. In otherembodiments, the two polypeptides that are covalently linked are part ofa continuous polypeptide chain, but are not directly fused to each other(i.e., the two polypeptides may be separated by one or more amino acids,a linker or another polypeptide). The term “covalently linked” does notimply a specific orientation of the two or more polypeptides that arefused to each other.

IL-15 is a 14-15 KD cytokine with structural similarity to IL-2. IL-15is also known as MGC9721. A variety of cell types constitutively produceIL-15 mRNA, and these include monocytes, macrophages, DCs,keratinocytes, epidermal skin cells, fibroblasts, various epithelialcells, bone marrow stromal cells, and nerve cells. In addition, IL-15mRNA is also produced in kidney, placenta, lung, heart, skeletal muscle,and brain tissues. However, only monocytes, DCs, epithelial cells, bonemarrow stromal cells, fibroblasts, and very few other cells and tissuessecrete detectable levels of IL-15. IL-15 and IL-2 are found to bind thesame hematopoietin subunits and share many biological activities. IL-15regulates T and NK cell activation and proliferation, and the number ofCD8⁺ memory cells is affected by a balance between IL-15 and IL-2. Inembodiments, the IL-15 or IL-15 derivative disclosed herein has at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of the activity ofhuman IL-15. IL-15 may be a mammalian IL-15, preferably a primate IL-15,and more preferably a human IL-15. The human IL-15 (Accession numberNP_000576) amino acids sequence is provided as SEQ ID NO:212.

The term “IL-15 derivative” refers to a polypeptide having at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity with SEQ ID NO:212, themature form of human I1-15. Techniques for making such derivatives areknown in the art. In some embodiments, the IL-15 or IL-15 derivativesequence may comprise one or more amino acid substitutions. In someembodiments, the amino acid substitutions are located at positions 1, 4,8, 30, 45, 61, 64, 65, and/or 108 of IL-15 (residue numbers arereferring to the mature form of the IL-15 protein). In some embodiments,the amino acid substitutions are located at position 45, 65, and/or 108of IL-15. In some embodiments the amino acid substitutions in IL-15 areN1D, N4D, D8N, D30N, D61N, E64Q, L45A, N65S/A/D/K, and/or Q108S/E. Insome embodiments, the amino acid substitutions in IL-15 are L45A,N65S/A/D/K, and/or Q108S. In embodiments, a fusion protein disclosedherein comprises an IL-15 polypeptide comprising one of the followingamino acid substitutions: N65S, N65A, or N65D. In embodiments, a fusionprotein disclosed herein comprises an IL-15 polypeptide comprising oneor more of the following amino acid substitutions: L45A, N65S, N65A, orN65D, and Q108S.

The amino acid sequence of human IL-15Rα isoform 1 precursor (Accessionnumber NP_002180) is provided in SEQ ID NO:213. The IL-15 receptor,i.e., IL-15 receptor complex, specifically binds IL-15 with highaffinity and consists of a unique interleukin 15 receptor αsubunit,IL-2/IL-15Rβ, and the common 7-chain/IL-2RT subunit. IL-15Rα isexpressed by mitogen-activated macrophages, NK cells, and CD4⁺ and CD8⁺T cells. The human IL-15Rα consists of seven exons, and alternative mRNAsplicing may result in eight molecular IL-15Rα isoforms with differentextra or intracellular domains. Full-length isoforms consist of anextracellular portion containing a conserved protein binding motif(sushi domain), a trans-membrane domain, and an intracellular tail.

As used herein, the term “sushi domain” of IL-15Rα refers to a domainbeginning at the first cysteine residue (C1) after the signal peptide ofIL-15Rα and ending at the fourth cysteine residue (C4) after said signalpeptide. The sushi domain corresponding to a portion of theextracellular region of IL-15Rα is involved in binding to IL-15. Thesushi domain in the present disclosure has at at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or at least 90% of the activity of the sushi domainof the human IL-15Rα chain.

The amino acid sequence of human IL-15Rα isoform 1 precursor is providedin SEQ ID NO:213 (this sequence includes the signal sequence). The sushidomain amino acid sequence of human IL-15Rα is provided in SEQ ID NO:214.

The term “IL-15Rα sushi domain derivative” or “IL-15Rα sushi domainvariant” refers to a polypeptide having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity with the sequence of the human IL-15Rα sushidomain (SEQ ID NO:214). Techniques for making such derivatives orvariants are known in the art. All such derivatives comprise the fourcysteine residues of the sushi domain of IL-15Rα. In some suchderivatives, naturally occurring amino acids may be replaced bychemically modified amino acids to alter the polypeptide half-life. Insome embodiments, the IL-15Rα sushi domain or IL-15Rα sushi domainderivative sequence may comprise one or more amino acid substitutions.In some embodiments, the amino acid substitution is located at position60 of IL-15Rα (numbering of the IL-15Rα residues include the signalpeptide, see, e.g., SEQ ID NO:213). In some embodiments, the amino acidsubstitution in the IL-15Rα sushi domain is N60A (i.e., the asparagine(N) at amino acid 60 is substituted with alanine (A)).

IL-15 binds with high affinity to IL-15Rα, which then associates with anIL-15Rβ/γc complex expressed by the same target cell (cis-presentation).IL-15Rα is also known to trans-present IL-15 with high affinity to adifferent target cell expressing the IL-15Rβ/γ(c) complex(trans-presentation). IL-15 cis- and trans-presentation mechanisms leadto different dynamics of receptor activation and signal transduction,with cis-presentation inducing fast and transient responses, andtrans-presentation inducing slower, more persistent ones.

In some embodiments, the fusion proteins disclosed herein comprise astimulatory domain, which comprises a hybrid domain comprising the sushidomain of the IL-15Rα chain attached to IL-15 by a linker (including,but not limited to, the linker of SEQ ID NO: 215). In certainembodiments, the stimulatory domain disclosed herein is covalentlylinked to an anti-PD-1 antibody or antigen-binding fragment thereofdisclosed herein via a second linker (including, but not limited to, thelinker of SEQ ID NO: 216). In other embodiments, the stimulatory domainexemplified herein is directly fused to an anti PD-1 antibody orantigen-binding fragment disclosed herein.

In one embodiment, provided is an “N-terminal fusion protein,” wherein astimulatory domain is linked directly or via a linker (e.g., via one ormore peptides) to the N-terminus of an anti-PD1 binding portion of thefusion protein (e.g., the N-terminus of a heavy chain of an anti-PD-1antibody disclosed herein).

N-terminal fusion proteins include but are not limited to the following(exemplary) fusion proteins (components recited from N- to C-terminus,“-” indicated direct covalent linkage or linkage via a linker (e.g., viaone or more peptides):

-   -   (1) (IL-15 or derivative thereof)—(heavy chain of anti-PD-1        antibody or antigen binding fragment thereof)    -   (2) (IL-15 or derivative thereof)—(light chain of anti-PD-1        antibody or antigen binding fragment thereof)    -   (3) (IL-15Rα polypeptide comprising the IL-15Rα sushi domain or        a derivative thereof)—(IL-15 or derivative thereof)—(heavy chain        of anti-PD-1 antibody or antigen binding fragment thereof)    -   (4) (IL-15Rα polypeptide comprising the IL-15Rα sushi domain or        a derivative thereof)—(IL-15 or derivative thereof)—(light chain        of anti-PD-1 antibody or antigen binding fragment thereof)    -   (5) (IL-15 or derivative thereof)—(IL-15Rα polypeptide        comprising the IL-15Rα sushi domain or a derivative        thereof)—(heavy chain of anti-PD-1 antibody or antigen binding        fragment thereof)    -   (6) (IL-15 or derivative thereof)—(IL-15Rα polypeptide        comprising the IL-15Rα sushi domain or a derivative        thereof)—(light chain of anti-PD-1 antibody or antigen binding        fragment thereof)

N-terminal fusion proteins may comprise more than one stimulatorydomain.

In one embodiment, provided is a fusion protein, wherein the stimulatorydomain is covalently linked to the N-terminus of one (and only one) ofthe heavy chains of the anti-PD-1 antibody or antigen binding fragmentthereof. An N-terminal fusion protein comprising a single stimulatorydomain is referred to as a “i-N-terminal fusion protein.”

Provided herein is a fusion protein comprising (1) a stimulatory domain(2) a first heavy chain of an anti-PD-1 antibody or antigen bindingfragment thereof, wherein the stimulatory domain is covalently linked tothe N-terminus of the first heavy chain, and (3) a second heavy chain ofan anti-PD-1 antibody or antigen binding fragment thereof, wherein thesecond heavy chain is not linked to a stimulatory domain. In someembodiments, the stimulatory domain comprises (i) IL-15 or a derivativethereof, or (ii) an IL-15Rα polypeptide comprising the IL-15Rα sushidomain or a derivative thereof, or (iii) both.

In one embodiment, provided is a fusion protein, wherein a stimulatorydomain is covalently linked to the N-termini of both of the heavy chainsof the anti-PD-1 antibody or antigen binding fragment thereof. AnN-terminal fusion protein comprising two stimulatory domains, bothlinked to different antibody chains, is referred to as a “2-N-terminalfusion protein.”

In one embodiment, provided is an “C-terminal fusion protein,” wherein astimulatory domain is linked directly or via a linker (e.g., via one ormore peptides) to the C-terminus of an anti-PD1 binding portion of thefusion protein (e.g., the C-terminus of a heavy chain of an anti-PD-1antibody disclosed herein).

C-terminal fusion proteins include but are not limited to the following(exemplary) fusion proteins (components recited from N- to C-terminus,“-” indicated direct covalent linkage or linkage via a linker (e.g., viaone or more peptides):

-   -   (1) (heavy chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15 or derivative thereof)    -   (2) (light chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15 or derivative thereof)    -   (3) (heavy chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15Rα polypeptide comprising the IL-15Rα        sushi domain or a derivative thereof)—(IL-15 or derivative        thereof)    -   (4) (light chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15Rα polypeptide comprising the IL-15Rα        sushi domain or a derivative thereof)—(IL-15 or derivative        thereof)    -   (5) (heavy chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15 or derivative thereof)—(an IL-15Rα        polypeptide comprising the IL-15Rα sushi domain or a derivative        thereof)    -   (6) (light chain of anti-PD-1 antibody or antigen binding        fragment thereof)—(IL-15 or derivative thereof)—(an IL-15Rα        polypeptide comprising the IL-15Rα sushi domain or a derivative        thereof)

C-terminal fusion proteins may comprise more than one stimulatorydomain.

In one embodiment, provided is a fusion protein, wherein the stimulatorydomain is covalently linked to the C-terminus one (and only one) of theheavy chains of the anti-PD-1 antibody or antigen binding fragmentthereof. A C-terminal fusion protein comprising a single stimulatorydomain is referred to as a “i-C-terminal fusion protein.”

Provided herein is a fusion protein comprising (1) a stimulatory domain,(2) a first heavy chain of an anti-PD-1 antibody or antigen bindingfragment thereof, wherein the stimulatory domain is covalently linked tothe C-terminus of the first heavy chain, and (3) a second heavy chain ofan anti-PD-1 antibody or antigen binding fragment thereof, wherein thesecond heavy chain is not linked to a stimulatory domain. In someembodiments, the stimulatory domain comprises (i) IL-15 or a derivativethereof, or (ii) an IL-15Rα polypeptide comprising the IL-15Rα sushidomain or a derivative thereof, or (iii) both.

In one embodiment, provided is a fusion protein, wherein a stimulatorydomain is covalently linked to the C-termini of both of the heavy chainsof the anti-PD-1 antibody or antigen binding fragment thereof. AC-terminal fusion protein comprising two stimulatory domains, bothlinked to different antibody chains, is referred to as a “2-C-terminalfusion protein.”

FIG. 21 depicts schematic diagrams of exemplary fusion proteins asdescribed herein.

Therapeutic levels of IL-2 are associated with high level of toxicityupon binding to the IL-2 receptor. Similarly, using therapeutic levelsof IL-15 can also cause toxicity since IL-15 shares two receptorsubunits with IL-2. The N-terminal fusion proteins, however, demonstratereduced toxity compared to the C-terminal fusion proteins because whenpresented in cis position, the N-terminal IL-15 fusion proteins canselectively bind to tumor infiltrating lymphocyte (TIL) while showing alower binding to peripheral T cells. When presented in cis position,N-terminal fusion proteins may bind to IL-15Rβγ and PD-1 simultaneouslywhile reducing the IL-15Rβ/γ binding, allowing the N-terminal fusionproteins to selectively bind to TILs and promoting TIL proliferation. Incontrast, C-terminal fusion proteins may bind to both peripheral T cellsand TIL equally and may show higher toxicity than the N-terminal fusionproteins. (FIG. 25 ). 1N-terminal fusion proteins show reduced toxicitycompared to an N-terminal fusion protein comprising two stimulatorydomains covalently linked to an anti-PD-1 antibody disclosed herein(2N-terminal fusion protein). Mutated N-terminal fusion proteins show afurther reduction in toxicity due to even lower binding to peripheral Tcells.

In one aspect, the disclosure relates to a fusion protein comprising:

-   -   (i) an anti-PD-1 antibody, or an antigen-binding fragment        thereof, wherein the anti-PD-1 antibody or antigen-binding        fragment thereof comprises a heavy chain variable region and a        light chain variable region, wherein each of the heavy chain and        the light chain variable regions comprise a CDR1, CDR2, and        CDR3, and wherein the antibody heavy chain comprises a constant        region comprising three constant domains CH1, CH2, and CH3;    -   (ii) an interleukin 15 (IL-15) polypeptide comprising an amino        acid sequence of SEQ ID NO: 212, or an amino acid sequence that        is at least 90% or at least 95% identical to SEQ ID NO: 212;    -   (iii) an IL-15Rα sushi domain polypeptide comprising an amino        acid sequence of SEQ ID NO: 214, or an amino acid sequence that        is at least 90% or at least 95% identical to SEQ ID NO: 214; and    -   (iv) a first linker polypeptide joining the IL-15Rα sushi domain        polypeptide and the IL-15 polypeptide.

The anti-PD-1 antibody, or antigen-binding fragment thereof, portion ofthe fusion protein can be any that specifically bind PD-1, includingthose comprising the corresponding variable regions or CDRs provided inFIG. 1 , or otherwise described herein. In embodiments, the fusionprotein comprises an antibody, or antigen-binding fragment thereof,wherein the CDRs of the heavy and light chain variable regions comprisethe following sequences: the sequence CDR1H comprises the sequence ofSEQ ID NO: 21;

-   -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23;    -   the sequence of CDR1L comprises the sequence        RX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₈WX₁₉X₂₀ (SEQ ID NO:135);        -   wherein X₁₃ is A or V;        -   wherein X₁₄ is S or G;        -   wherein X₁₅ is Q, E, or R;        -   wherein X₁₆ is G, S, D or N;        -   wherein X₁₇ is G, S, or N;        -   wherein X₁₈ is S, I, R, T, K, P, N, H, or V;        -   wherein X₁₉ is L or V; and        -   wherein X₂₀ is G or A; and    -   the sequence of CDR2L comprises the sequence X₂₁AX₂₂X₂₃X₂₄X₂₃X₂₆        (SEQ ID NO: 136); and        -   wherein X₂₁ is S, D, E, or A;        -   wherein X₂₂ is S or K;        -   wherein X₂₃ is S, N, T, R, or D;        -   wherein X₂₄ is L or V;        -   wherein X₂₅ is Q, E, or H; and        -   wherein X₂₆ is S, N, A, R, P, or T; and    -   the sequence of CDR3L comprises the sequence QQX₂₇X₂₈SFPX₂₉X₃₀        (SEQ ID NO: 137);        -   wherein X₂₇ is A or G;        -   wherein X₂₈ is N, D, or Y;        -   wherein X₂₉ is F or L; and        -   wherein X₃₀ is A or T.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy andlight chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

In further embodiments, the fusion protein comprises an anti-PD-1antibody, or antigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   the light chain variable region comprises SEQ ID NO: 98, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        98.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequences of CDR1H, CDR2H and CDR3H comprise respectively        SEQ ID NOS: 21, 22, and 23; and    -   the sequences of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 45, 46, and 47; SEQ ID NOS: 66, 67, and 68; SEQ ID        NOS: 70, 67, and 71; SEQ ID NOS: 73, 74, and 75; SEQ ID NOS: 77,        78, and 47; SEQ ID NOS: 80, 81, and 82; SEQ ID NOS: 77, 78, and        84; SEQ ID NOS: 77, 86, and 47; SEQ ID NOS: 88, 89, and 47; SEQ        ID NOS: 66, 67, and 47; SEQ ID NOS: 80, 92, and 75; SEQ ID NOS:        80, 94, and 71; SEQ ID NOS: 99, 100, and 47; SEQ ID NOS: 102,        103, and 104; SEQ ID NOS: 106, 103, and 47; SEQ ID NOS: 108,        103, and 47; SEQ ID NOS: 110, 111, and 75; SEQ ID NOS: 77, 103,        and 113; SEQ ID NOS: 77, 111, and 47; SEQ ID NOS: 116, 67, and        47; SEQ ID NOS: 118, 119, and 47; SEQ ID NOS: 80, 78, and 47;        SEQ ID NOS: 122, 103 and 47; SEQ ID NOS: 124, 125, and 75; SEQ        ID NOS: 127, 38, and 68; SEQ ID NOS: 129, 130, and 47; or SEQ ID        NOS: 132, 133, and 75.

In further embodiments, the fusion protein comprises an anti-PD-1antibody, or antigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   the light chain variable region comprises SEQ ID NO: 48, 69, 72,        76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115,        117, 120, 121, 123, 126, 128, 131, or 134, or a sequence that is        at least 90%, at least 95%, at least 96%, at least 97%, at least        98%, or at least 99% identical to any one of SEQ ID NO: 48, 69,        72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114,        115, 117, 120, 121, 123, 126, 128, 131, or 134.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy andlight chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence        RSSX₁SLLX₂SNGX₃X₄YLD (SEQ ID NO:62),        -   wherein X₁ is Q or E;        -   wherein X₂ is H or Y;        -   wherein X₃ is Y or N; and        -   wherein X₄ is T or N;    -   the sequence of CDR2L comprises the sequence X₅X₆SX₇X₈X₉X₁₀ (SEQ        ID NO: 63),        -   wherein X₅ is L, Q or E;        -   wherein X₆ is S, A, or V;        -   wherein X₇ is H, N, T, or S;        -   wherein X₈ is R or L;        -   wherein X₉ is G, A, or H; and        -   wherein X₁₀ is S or T; and    -   the sequence of CDR3L comprises the sequence MQGX₁₁X₁₂WPYT (SEQ        ID NO: 64),        -   wherein X₁₁ is A, T, or S; and        -   wherein X₁₂ is H or R.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 41;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 49;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 50.

In further embodiments, the fusion protein comprises an anti-PD-1antibody, or antigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   the light chain variable region comprises SEQ ID NO: 51, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        51.

In embodiments, the fusion protein comprises an anti-PD-1 antibody, orantigen-binding fragment thereof, wherein the CDRs of the heavy chainand light chain variable regions comprise the following sequences:

-   -   the sequence of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17,        18, and 19, respectively; and    -   the sequence of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 41, 42, and 43; SEQ ID NOS: 41, 52, and 53; SEQ ID        NOS: 41, 55, and 56; or SEQ ID NOS: 58, 59, and 60.

In further embodiments the fusion protein comprises an anti-PD-1antibody, or antigen-binding fragment thereof, wherein:

-   -   the heavy chain variable region comprises SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   the light chain variable region comprises SEQ ID NO: 44, 54, 57,        or 61, or a sequence that is at least 90%, at least 95%, at        least 96%, at least 97%, at least 98%, or at least 99% identical        to any one of SEQ ID NO: 44, 54, 57, or 61.

Provided herein is a fusion protein that comprises a sequence describedin Tables 24-27.

Provided herein is a fusion protein that comprises a heavy chaincomprising a sequence selected from the group consisting of SEQ ID NOS:174-184, 187-190, 193-199, 206-211, 224-243. Provided herein is a fusionprotein that comprises a light chain comprising a sequence selected fromthe group consisting of SEQ ID NOS: 170, 173, 186, 192, and 205.

Provided herein is a fusion protein that comprises a light chain and aheavy/heavy chain combination recited in Table 26.

Provided herein is a fusion protein that comprises a light chain and oneor more heavy chains recited in Table 27.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:224;    -   (2) a first heavy chain sequence comprising SEQ ID NO:221 and a        second heavy chain sequence comprising SEQ ID NO:225;    -   (3) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:226;    -   (4) a first heavy chain sequence comprising SEQ ID NO:223 and a        second heavy chain sequence comprising SEQ ID NO:227.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:228;    -   (2) a first heavy chain sequence comprising SEQ ID NO:221 and a        second heavy chain sequence comprising SEQ ID NO:229;    -   (3) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:230;    -   (4) a first heavy chain sequence comprising SEQ ID NO:223 and a        second heavy chain sequence comprising SEQ ID NO:231;    -   (5) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:236;    -   (6) a first heavy chain sequence comprising SEQ ID NO:221 and a        second heavy chain sequence comprising SEQ ID NO:237;    -   (7) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:238;    -   (8) a first heavy chain sequence comprising SEQ ID NO:223 and a        second heavy chain sequence comprising SEQ ID NO:239;    -   (9) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:241; or    -   (10) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:242.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:232;    -   (2) a first heavy chain sequence comprising SEQ ID NO:221 and a        second heavy chain sequence comprising SEQ ID NO:233;    -   (3) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:234;    -   (4) a first heavy chain sequence comprising SEQ ID NO:223 and a        second heavy chain sequence comprising SEQ ID NO:235; or    -   (5) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:243.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:224; or a first        heavy chain sequence comprising SEQ ID NO:221 and a second heavy        chain sequence comprising SEQ ID NO:225; or    -   (2) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:226; or a first        heavy chain sequence comprising SEQ ID NO:223 and a second heavy        chain sequence comprising SEQ ID NO:227.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:228; or a first        heavy chain sequence comprising SEQ ID NO:221 and a second heavy        chain sequence comprising SEQ ID NO:229;    -   (2) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:230; or a first        heavy chain sequence comprising SEQ ID NO:223 and a second heavy        chain sequence comprising SEQ ID NO:231;    -   (3) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:236; or a first        heavy chain sequence comprising SEQ ID NO:221 and a second heavy        chain sequence comprising SEQ ID NO:237;    -   (4) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:238; or a first        heavy chain sequence comprising SEQ ID NO:223 and a second heavy        chain sequence comprising SEQ ID NO:239;    -   (5) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:241; or    -   (6) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:242.

In embodiments, provided is a fusion protein comprising a light chainsequence comprising SEQ ID NO:219 and/or SEQ ID NO:98 and:

-   -   (1) a first heavy chain sequence comprising SEQ ID NO:220 and a        second heavy chain sequence comprising SEQ ID NO:232; or a first        heavy chain sequence comprising SEQ ID NO:221 and a second heavy        chain sequence comprising SEQ ID NO:233;    -   (2) a first heavy chain sequence comprising SEQ ID NO:222 and a        second heavy chain sequence comprising SEQ ID NO:234; or a first        heavy chain sequence comprising SEQ ID NO:223 and a second heavy        chain sequence comprising SEQ ID NO:235; or    -   (3) a first heavy chain sequence comprising SEQ ID NO:240 and a        second heavy chain sequence comprising SEQ ID NO:243.

Antibodies and Antigen-Binding Fragments Thereof

The term antibody is used here in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, bispecific antibodies, humanizedantibodies, single chain antibodies, chimeric antibodies, syntheticantibodies, recombinant antibodies, hybrid antibodies, mutagenizedantibodies and grafted antibodies (grafted antibodies), bispecificantibodies, a specific antibody portion (e.g., a domain antibody), aswell as any antigen-binding portion thereof that competes with an intactantibody for specific binding, an antigen-binding portion thereof (e.g.,paratopes, CDRs), and any other modified conformations of theimmunoglobulin molecule comprising the antigen recognition site so longas they exhibit the desired biological activity and specificity.Accordingly, an antibody is an immunoglobulin molecule or fragment orderivative thereof including any polypeptide comprising anantigen-binding site, capable of specifically binding to a targetthrough at least one antigen recognition site located in the variableregion of the immunoglobulin molecule. The disclosed antibody can bemurine, rat, human, or any other origin (including chimeric or humanizedantibodies).

In certain embodiments, the framework regions of the antibody (orantigen-binding fragment thereof) may be identical to human germlinesequences or may be naturally or artificially modified.

In one preferred embodiment, the disclosed antibody structures belong tothe IgG class of immunoglobulin molecules. A standard IgG immunoglobulinmolecule comprises two identical light chain polypeptides, and twoidentical heavy chain polypeptides. The molecular weight of the lightchain polypeptide is around 23,000 Daltons and the molecular weight ofthe heavy chain polypeptide varies between 53,000-70,000 Daltons. Thefour chains are typically joined by disulfide bonds in a “Y”configuration.

Two heavy chains (HC) and two light chains (LC) of an immunoglobulinmolecule are covalently bonded to each other, and the end portions ofthe two heavy chains are bonded to each other by covalent disulfidelinkages or non-covalent linkages when the immunoglobulins are generatedby either hybridomas, B cells, or genetically engineered host cells. Thelight and heavy chains both contain regions of structural and functionalhomology. The term “variable” and “constant” are used functionally. Eachheavy chain is comprised of a heavy chain variable region (“HCVR” or“VH”) and a heavy chain constant region (comprised of domains CH1, CH2,and CH3). Each light chain is comprised of a light chain variable region(“LCVR or “VL”) and a light chain constant region (CL). The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The variable region allows the antibody to recognize and specificallybind epitopes located on antigens. The variable domains of both thelight (VL) and heavy (VH) chain portions determine antigen recognitionand specificity. The antigen-binding site of an antibody is comprised ofthe VL domain and VH domain, or a subset of the CDRs. More specifically,the antigen-binding site is defined by one, two, or three CDRs on eachof the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2,and CDR-L3). Conversely, the constant domains of the light chain (CL)and the heavy chain (CH1, CH2, or CH3) confer biological properties suchas secretion, transplacental mobility, Fc receptor binding, complementbinding, and the like. By convention, the numbering of the constantregion domains increases as they become more distal from theantigen-binding site or amino-terminus of the antibody.

As used herein, the term “Complementarity Determining Regions” (CDRs)refers to portions of an antibody variable domain that are (typically)involved in antigen binding. Each variable region has threenon-consecutive CDRs, known as CDR1, CDR2, and CDR3. The CDRs areseparated by structurally conserved regions called framework regions(FR-1, -2, -3, and -4) that form a “core” β-sheet structure displayingthese loops on the surface of the variable domain. The six CDRs presentin each antigen-binding domain are short, non-contiguous sequences ofamino acids that are specifically positioned to form the antigen-bindingdomain as the antibody assumes its three dimensional configuration in anaqueous environment. The length and composition of the CDR sequences arehighly variable, especially in the CDR3. The remainder of the aminoacids located in the antigen-binding domains or the “framework” regions,show less inter-molecular variability. The antigen-binding domain formedby the positioned CDRs defines a surface complementary to the epitope onthe immunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. Each CDRcan comprise amino acid residues from a CDR as defined by e.g. Kabat(i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the lightchain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in theheavy chain variable domain (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1987, 1991)). Each CDR can alsocomprise amino acid residues from a “hypervariable loop” (i.e., aboutresidues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain (Chothia & Lesk 196 J. Mol. Biol. 901 (1987)). Insome instances, a CDR can include amino acids from both a CDR regiondefined according to Kabat and a hypervariable loop. The Kabat numberingmay not always correspond to the linear numbering on the amino acidresidues due to a shortening of, or insertion into, a structuralcomponent, whether framework or CDR, of the basic variable domainstructure. The correct Kabat numbering of residues may be determined fora given antibody or antigen-binding fragment thereof by alignment ofresidues of homology in the sequence of the antibody or antigen-bindingfragment thereof with a “standard” Kabat numbered sequence or be definedaccording to ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev.Comp. Immunol., 27, 55-77 (2003)).

As used herein, the term “antigen-binding portion” or “antigen-bindingfragment” may be a fragment comprising a Fab, Fab′, F(ab′)2, Fd, Fv,domain antibodies (dAbs such as shark and camel antibodies), ScFv, amaxibody, a minibody, a nanobody, an intrabody, a diabody, a triabody, atetrabody, a v-NAR and a bis-scFv, or a polypeptide that contain atleast certain portions of an immunoglobulin sufficient to conferspecific antigen-binding to the polypeptide.

The antibody may be any class of antibody, such as IgG, IgA, or IgM (ora subclass thereof), and the antibody need not be of any particularclass, and any of the immunoglobulin molecules comprising the antigenrecognition site of the required specificity, other modifiedconfigurations (including glycosylation variants of antibodies, aminoacid sequence variants of antibodies, and covalently modifiedantibodies) can be encompassed. Modified versions of each of theseclasses and isotypes are known to a person skilled in the art,accordingly, are within the scope of the instant disclosure.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a Fab fragment, which comprises or consistessentially of a variable (VL) and constant (CL) domain of the lightchain and a variable domain (VH) and the first constant domain (CH1) ofthe heavy chain.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a Fab′ fragment, which refers to a Fab fragmenthaving one or more cysteine residues at the C-terminus of the CH1domain.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is an Fd fragment comprising or consisting essentiallyof VH and CH1 domains.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is an Fd′ fragment comprising VH and CH1 domains andone or more cysteine residues at the C-terminus of the CH1 domain.

Single-chain Fv or scFv antibody fragments comprise or consistessentially of the VH and VL domains of an antibody, such that thesedomains are present in a single polypeptide chain. Generally, an Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains, which allows the scFv to form the desired structure forantigen-binding. Accordingly, in some embodiments of the aspectsdescribed herein, the anti-PD-1 antibody fragment is a Fv fragmentcomprising or consisting essentially of the VL and VH domains of asingle arm of an antibody.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a diabody comprising two antigen-binding sites,comprising a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a dAb fragment comprising or consisting essentiallyof a VH domain.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a F(ab′)2 fragment, which comprises a bivalentfragment comprising two Fab′ fragments linked by a disulfide bridge atthe hinge region.

In some embodiments of the aspects described herein, the anti-PD-1antibody fragment is a linear antibody comprising a pair of tandem Fdsegments (VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen-binding regions.

A person skilled in the arts can use various techniques that have beendeveloped and are available for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies. However, F(ab′)2 fragments can be isolated directlyfrom recombinant host cell culture. Other techniques for the productionof antibody fragments will be apparent to the skilled practitioner. Inother embodiments, the antibody fragment of choice is a single chain Fvfragment (scFv). See, for example, WO 93/16185. Alternatively, thesefragments can also be produced directly by recombinant host cells. Forexample, antibody fragments can be isolated from the antibody phagelibraries discussed herein. In another approach, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)2 fragments (Carter et al., 1992).

In one embodiment, the antibody is a bispecific antibody comprising acomplementary region that binds PD-1.

Contemplated antibodies or antigen-binding fragments may have all typesof constant regions, including IgM, IgG, IgD, and IgE, and any isotype,including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the isotype ishuman IgG1. In another embodiment, the human isotype IgG4 is used. Lightchain constant regions can be X or u. The antibody or antigen-bindingfragment thereof may comprise sequences from more than one class orisotype.

Anti-PD-1 Antibodies and PD-1 Binding Fragments Thereof

The disclosure describes antibodies that bind to PD-1, andantigen-binding fragments thereof that bind to PD-1, as well as fusionproteins comprising such anti-PD-1 antibodies or antigen-bindingfragments thereof. The term “PD-1” refers to programmed death-1 protein(also known as CD279), a T cell co-inhibitor. The term PD-1 encompassesrecombinant PD-1 and/or a fragment thereof. The term also includes PD-1or a fragment thereof coupled to, for example, mouse or human Fc,histidine tag, and/or a signal sequence. The term may further encompassa fusion protein comprising PD-1. The amino acid sequence of full-lengthPD-1 is provided in GenBank as accession number NP_005009.2. The ligandsfor PD-1 include PD-L1 and PD-L2. The amino acid sequence of full-lengthPD-L1 is provided in GenBank as accession number NP_054862.1. The aminoacid sequence of full-length PD-L2 is provided in GenBank as accessionnumber NP_079515.2.

In embodiments, the anti-PD-1 antibody or antigen binding fragmentthereof specifically binds to PD-1 and antagonizes PD-1 mediated immunesuppression. The anti-PD-1 antibodies and antigen-binding fragmentsthereof disclosed herein may interrupt, inhibit, or reduce PD-1biological activity including downstream events mediated by PD-1. Theanti-PD-1 antibodies and antigen-binding fragments thereof disclosedherein may exhibit any one or more of the following features: (a)binding to PD-1 and blocking of downstream signaling events; (b)blocking PD-L1-binding to PD-1; (c) increasing T cell proliferation; (d)upregulating the T cell-mediated immune response; (e) stimulating TNFsecretion; (f) reducing inhibitory signal transduction through PD-1;and/or (g) stimulating IFNγ secretion. The anti-PD-1 antibodies andantigen-binding fragments thereof disclosed herein exhibit potentbinding and inhibitory activities and are useful for therapeutic anddiagnostics uses.

In one aspect, the disclosure provides antibodies and antigen-bindingfragments thereof that bind to PD-1. In certain embodiments, thedisclosure provides bispecific antibodies and binding proteins that bindspecifically to PD-1 and at least one other molecule.

In another aspect the disclosure provides an antibody, or anantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment thereof binds to PD-1, and wherein the antibodyor antigen-binding fragment thereof comprises a heavy chain variableregion and a light chain variable region wherein each of the heavy chainand the light chain variable regions comprise a CDR1, CDR2, and CDR3 andwherein the antibody heavy chain comprises a constant region comprisingthree constant domains CH1, CH2, and CH3.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23;    -   the sequence of CDR1L comprises the sequence        RX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₅WX₁₉X₂₀ (SEQ ID NO:135);        -   wherein X₁₃ is A or V;        -   wherein X₁₄ is S or G;        -   wherein X₁₅ is Q, E, or R;        -   wherein X₁₆ is G, S, D or N;        -   wherein X₁₇ is G, S, or N;        -   wherein X₁₈ is S, I, R, T, K, P, N, H, or V;        -   wherein X₁₉ is L or V; and        -   wherein X₂₀ is G or A; and    -   the sequence of CDR2L comprises the sequence X₂₁AX₂₂X₂₃X₂₄X₂₃X₂₆        (SEQ ID NO: 136); and        -   wherein X₂₁ is S, D, E, or A;        -   wherein X₂₂ is S or K;        -   wherein X₂₃ is S, N, T, R, or D;        -   wherein X₂₄ is L or V;        -   wherein X₂₅ is Q, E, or H; and        -   wherein X₂₆ is S, N, A, R, P, or T; and    -   the sequence of CDR3L comprises the sequence QQX₂₇X₂₈SFPX₂₉X₃₀        (SEQ ID NO: 137);        -   wherein X₂₇ is A or G;        -   wherein X₂₈ is N, D, or Y;        -   wherein X₂₉ is F or L; and        -   wherein X₃₀ is A or T.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 21;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 22;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 23    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 96;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 82.

In embodiments, the antibody, or antigen-binding fragment thereof,comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   a light chain variable region comprising SEQ ID NO: 98, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        98.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequences of CDR1H, CDR2H and CDR3H comprise respectively        SEQ ID NOS: 21, 22, and 23; and    -   the sequences of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 45, 46, and 47; SEQ ID NOS: 66, 67, and 68; SEQ ID        NOS: 70, 67, and 71; SEQ ID NOS: 73, 74, and 75; SEQ ID NOS: 77,        78, and 47; SEQ ID NOS: 80, 81, and 82; SEQ ID NOS: 77, 78, and        84; SEQ ID NOS: 77, 86, and 47; SEQ ID NOS: 88, 89, and 47; SEQ        ID NOS: 66, 67, and 47; SEQ ID NOS: 80, 92, and 75; SEQ ID NOS:        80, 94, and 71; SEQ ID NOS: 99, 100, and 47; SEQ ID NOS: 102,        103, and 104; SEQ ID NOS: 106, 103, and 47; SEQ ID NOS: 108,        103, and 47; SEQ ID NOS: 110, 111, and 75; SEQ ID NOS: 77, 103,        and 113; SEQ ID NOS: 77, 111, and 47; SEQ ID NOS: 116, 67, and        47; SEQ ID NOS: 118, 119, and 47; SEQ ID NOS: 80, 78, and 47;        SEQ ID NOS: 122, 103 and 47; SEQ ID NOS: 124, 125, and 75; SEQ        ID NOS: 127, 38, and 68; SEQ ID NOS: 129, 130, and 47; or SEQ ID        NOS: 132, 133, and 75.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 24, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        24; and    -   a light chain variable region comprising SEQ ID NO: 48, 69, 72,        76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114, 115,        117, 120, 121, 123, 126, 128, 131, or 134, or a sequence that is        at least 90%, at least 95%, at least 96%, at least 97%, at least        98%, or at least 99% identical to any one of SEQ ID NO: 48, 69,        72, 76, 79, 83, 85, 90, 91, 93, 101, 105, 107, 109, 112, 114,        115, 117, 120, 121, 123, 126, 128, 131, or 134.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence        RSSX₁SLLX₂SNGX₃X₄YLD (SEQ ID NO:62),        -   wherein X₁ is Q or E;        -   wherein X₂ is H or Y;        -   wherein X₃ is Y or N; and        -   wherein X₄ is T or N;    -   the sequence of CDR2L comprises the sequence X₅X₆SX₇X₈X₉X₁₀ (SEQ        ID NO: 63),        -   wherein X₅ is L, Q or E;        -   wherein X₆ is S, A, or V;        -   wherein X₇ is H, N, T, or S;        -   wherein X₈ is R or L;        -   wherein X₉ is G, A, or H; and        -   wherein X₁₀ is S or T; and    -   the sequence of CDR3L comprises the sequence MQGX₁₁X₁₂WPYT (SEQ        ID NO: 64),        -   wherein X₁₁ is A, T, or S; and        -   wherein X₁₂ is H or R.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H comprises the sequence of SEQ ID NO: 17;    -   the sequence of CDR2H comprises the sequence of SEQ ID NO: 18;    -   the sequence of CDR3H comprises the sequence of SEQ ID NO: 19;    -   the sequence of CDR1L comprises the sequence of SEQ ID NO: 41;    -   the sequence of CDR2L comprises the sequence of SEQ ID NO: 49;        and    -   the sequence of CDR3L comprises the sequence of SEQ ID NO: 50.

In embodiments, the anti-PD-1 antibody, or antigen-binding fragmentthereof, comprises:

-   -   a heavy chain variable region comprising SEQ ID NO: 20, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        20; and    -   a light chain variable region comprising SEQ ID NO: 51, or a        sequence that is at least 90%, at least 95%, at least 96%, at        least 97%, at least 98%, or at least 99% identical to SEQ ID NO:        51.

In embodiments, the CDRs of the anti-PD-1 antibody, or antigen-bindingfragment thereof, comprise the following sequences:

-   -   the sequence of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17,        18, and 19, respectively; and    -   the sequence of CDR1L, CDR2L and CDR3L comprise respectively,        SEQ ID NOS: 41, 42, and 43; SEQ ID NOS: 41, 52, and 53; SEQ ID        NOS: 41, 55, and 56; or SEQ ID NOS: 58, 59, and 60.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, and24, and wherein the sequence of the light chain variable regioncomprises a sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to a light chainvariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 28, 32, 36, 40, 44, 48, 51, 54, 57, 61, 65, 69, 72, 76,79, 83, 85, 88, 90, 91, 93, 95, 98, 101, 105, 107, 109, 112, 114, 115,117, 120, 121, 123, 126, 128, 131, 134, and 138.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 20, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the heavy chainvariable region amino acid sequence of SEQ ID NO: 65.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence SEQ IDNO: 20; and wherein the sequence of the light chain variable regioncomprises a sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to a light chainvariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 44, 51, 54, 57, and 61.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 20, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 51.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 20, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 54.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 20, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 57.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 20, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 61.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 138.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence SEQ IDNO: 20; and wherein the sequence of the light chain variable regioncomprises a sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to a light chainvariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 69, 72, 76, 79, 83, 85, 87, 90, 91, 93, 95, 98, 101, 105,107, 109, 112, 114, 115, 117, 120, 121, 123, 126, 128, 131, 134, and138.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 98.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 69.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 72.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 76.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 79.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 83.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 85.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 87.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 90.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 91.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 93.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 95.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 101.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 105.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 107.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 109.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 112.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 114.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 115.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 117.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 120.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 121.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 123.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 126.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 128.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 131.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 24, and wherein the sequence of the light chain variable regioncomprises the sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to the light chainvariable region amino acid sequence of SEQ ID NO: 134.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein the sequence of theheavy chain variable region comprises a sequence that is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the heavy chain variable region amino acid sequence of SEQID NO: 142, and wherein the sequence of the light chain variable regioncomprises a sequence that is at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to a light chainvariable region amino acid sequence selected from the group consistingof SEQ ID NOs: 146, 150, 153, 156, 160, 164, and 167.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 143;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 144;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 145.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 147;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 148;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 149.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 151;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 148;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 152.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 154;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 148;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 155.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 157;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 158;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 159.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 161;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 162;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 163.

In one embodiment, the disclosure provides an antibody orantigen-binding fragment thereof, which binds to PD-1, wherein theantibody or antigen-binding fragment comprises a heavy chain variableregion and a light chain variable region, wherein each of the heavychain and the light chain variable regions comprises a CDR1, CDR2, andCDR3, and wherein:

-   -   the sequence of CDR1H comprises the sequence, SEQ ID NO: 139;    -   the sequence of CDR2H comprises the sequence, SEQ ID NO: 140;    -   the sequence of CDR3H comprises the sequence, SEQ ID NO: 141;    -   the sequence of CDR1L comprises the sequence, SEQ ID NO: 157;    -   the sequence of CDR2L comprises the sequence, SEQ ID NO: 165;        and    -   the sequence of CDR3L comprises the sequence, SEQ ID NO: 166.

In addition to the above description, FIG. 1 shows the heavy and lightchain variable regions and associated CDR of antibodies disclosed hereinas does the accompanying sequence listing, which is incorporated byreference in its entirety. As shown in the Examples below, light chainshuffling of an anti-PD-1 antibody led to the identification of antibodyvariants that conferred substantial improvements in PD-1 bindingaffinity. Analysis of the variants revealed certain CDR positions atwhich amino acids remained relatively unchanged among the light chainshuffled antibodies and other CDR positions at which variation could beintroduced without abolishing PD-1 binding.

The anti-PD-1 antibodies and antigen-binding fragments thereof disclosedherein and the fusion proteins disclosed herein can have one or moreamino acid substitutions, deletions, insertions, and/or additions. Insome embodiments, one or more CDR residues of the anti-PD-1 antibodiesor antigen-binding fragments (or the fusion proteins comprising theanti-PD-1 antibodies or antigen-binding fragments) disclosed herein havebeen changed by amino acid substitution, deletion, insertion, and/oraddition. Amino acid substitutions can be conservative ornon-conservative substitutions. The present disclosure also includesanti-PD-1 antibodies, and antigen-binding fragments thereof (and thefusion proteins comprising the anti-PD-1 antibodies or antigen-bindingfragments), which are derived from the amino acid sequences disclosedherein, wherein one or more amino acids within one or more frameworkand/or CDR regions are mutated to the corresponding residue(s) of thegermline sequence from which the antibody was derived, or to thecorresponding residue(s) of another human germline sequence, or to aconservative amino acid substitution of the corresponding germlineresidue(s) (such sequence changes are referred to herein collectively as“germline mutations”). In certain embodiments, the anti-PD-1 antibodiesor binding fragments thereof (or the fusion proteins comprising theanti-PD-1 antibodies or antigen-binding fragments) comprise one or moreCDRs, or one or more variable domains with an amino acid sequence atleast 85% at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99%, identical to the CDR and/or variable domain sequences setforth in FIG. 1 .

Also provided herein variable heavy chain and variable light chainsequences as well as pairing thereof that are similar, but not identicalto the variable heavy chain and variable light chains disclosed in Table25 and pairings thereof. It will be evident that any of the frameworksdescribed herein can be utilized in combination with any of the CDRs andCDR motifs described herein. In some embodiments, the anti-PD-1 antibodyor antigen-binding fragment thereof utilizes a framework described inTable 25.

Also provided herein is a chimeric antigen receptor (CAR) comprisingone, two, three, four, five, or six CDRs of the anti-PD1 antibodies andantigen binding fragments disclosed herein. Also provided herein is CARcomprising the six CDRs of any one of the anti-PD1 antibodies andantigen binding fragments disclosed herein.

Disclosed is an immune cell expressing a CAR comprising one, two, three,four, five, or six CDRs of the anti-PD1 antibodies and antigen bindingfragments disclosed herein. Disclosed is an immune cell expressing a CARcomprising the six CDRs of any one of the anti-PD1 antibodies andantigen binding fragments disclosed herein. In some embodiments, theimmune cell is a T cell.

“Identity” refers to the number or percentage of identical positionsshared by two amino acid or nucleic acid sequences in optimally alignedsequences after considering number of gaps and the length of each gapthat were needed for the optimal alignment. “Substantially identical”means an amino acid sequence, which differs from the original sequenceonly by conservative amino acid substitutions, which do not destroy thefunction of the protein.

Also disclosed herein are anti-PD-1 antibodies or antigen-bindingfragments thereof or fusion proteins comprising an amino acid sequencethat is at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 98%, or at least 99% identical to an amino acid sequencedisclosed herein. Methods and computer programs for determining sequencesimilarity are publically available, including, but not limited to, theGCG program package (Devereux et al., Nucleic Acids Research 12: 387,1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403(1990), and the ALIGN program (version 2.0). The Smith Watermanalgorithm may also be used to determine similarity. The BLAST program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 athttp://www.ncbi.nlm.nih.gov/blast/). In comparing sequences, thesemethods account for various substitutions, deletions, and othermodifications.

In some embodiments of the aspects described herein, amino acid sequencemodification(s) of the antibodies or antigen-binding fragments thereofthat bind to PD-1 or the fusion proteins that bind to PD-1 describedherein are contemplated. Amino acid sequence variants of the anti-PD-1antibody or antigen-binding fragment thereof or the fusion proteins areprepared by introducing appropriate nucleotide changes into the nucleicacid encoding the anti-PD-1 antibody or antigen-binding fragment thereofor the fusion protein, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody or antigen-binding fragment thereof. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, if the final construct possesses the desired characteristics,e.g., binding specificity, inhibition of biological activity.

Amino acid substitutions can be made, in some cases, by selectingsubstitutions that do not differ significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, (b) the charge or hydrophobicity of the molecule at thetarget sit; or (c) the bulk of the side chain (conservative amino acidsubstitution variant. These variants have at least one amino acidresidue in the antibody or antigen-binding fragment thereof or fusionprotein replaced by a different residue that has similar side chainproperties. Amino acids can be grouped according to similarities in theproperties of their side chains (see Lehninger, BIOCHEMISTRY (2nd ed.,Worth Publishers, New York, 1975):

-   -   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe        (F), Trp (W), Met (M);    -   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr        (Y), Asn (N), Gln (Q);    -   (3) acidic: Asp (D), Glu (E);    -   (4) basic: Lys (K), Arg (R), His (H).

As such, a non-limiting example for a conservative amino acidsubstitution is one that replaces a non-polar amino acid with anothernon-polar amino acid.

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M);    -   (2) neutral hydrophilic: Ser (S), Thr (T), Cys (C), Asn (N), Gln        (Q);    -   (3) acidic: Asp (D), Glu (E);    -   (4) basic: Lys (K), Arg (R), His (H);    -   (5) residues that influence chain orientation: Gly (G), Pro (P);    -   (6) aromatic: Phe (F), Trp (W), Tyr (Y).

Substitutions made within these groups can be considered conservativesubstitutions. Examples of non-limiting substitutions include,substitution of valine for alanine, lysine for arginine, glutamine forasparagine, glutamic acid for aspartic acid, serine for cysteine,asparagine for glutamine, aspartic acid for glutamic acid, proline forglycine, arginine for histidine, leucine for isoleucine, isoleucine forleucine, arginine for lysine, leucine for methionine, leucine forphenylalanine, glycine for proline, threonine for serine, serine forthreonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/orleucine for valine.

Further contemplated are amino acid sequence insertions, which caninclude amino- and/or carboxyl-terminal fusions ranging in length fromone residue to polypeptides containing a hundred or more residues, aswell as intrasequence insertions of single or multiple amino acidresidues. Examples of terminal fusions include an antibody orantigen-binding fragment thereof with an N-terminal methionyl residue oran antibody or antigen-binding fragment thereof fused to a cytotoxicpolypeptide (or fusion proteins comprising such antibody orantigen-binding fragment thereof). Other examples of terminal fusions ofthe antibody or antigen-binding fragment thereof include the fusion tothe N- or C-terminus of the antibody or antigen-binding fragment thereofto an enzyme or a polypeptide which increases the serum half-life of theantibody or antigen-binding fragment thereof, such as, for example,biotin (or fusion proteins comprising such antibody or antigen-bindingfragment thereof).

Any cysteine residue not involved in maintaining the proper conformationof the antibodies or antigen-binding fragments thereof that bind to PD-1also can be substituted, for example with a serine or an alanine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

Conversely, cysteine bond(s) can be added to the anti-PD-1 antibody orantigen-binding fragment thereof to improve its stability (particularlywhere the anti-PD-1 antibody or antigen-binding fragment thereof is anantibody fragment such as an Fv fragment).

In some embodiments, the anti-PD-1 antibodies or antigen-bindingfragments thereof or the fusion protein comprising antibodies orantigen-binding fragments thereof describes have amino acid alterationsthat alter the original glycosylation pattern of the anti-PD-1 antibodyor antigen-binding fragment thereof. By “altering the originalglycosylation pattern” is meant deleting one or more carbohydratemoieties found in the antibody or antigen-binding fragment thereof,and/or adding one or more glycosylation sites that are not present inthe antibody or antigen-binding fragment thereof. Glycosylation ofantibodies is typically either N-linked or O-linked. N-linked refers tothe attachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, wherein X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone of the sugars N-aceylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine can also be used. The alteration canalso be made by the addition of, or substitution by, one or more serineor threonine residues to the sequence of the original antibody orantigen-binding fragment thereof (for O-linked glycosylation sites).

In some embodiments, the anti-PD-1 antibody or antigen-binding fragmentthereof may include a modification, including, but not limited toglycosylation, acetylation, pegylation, phosphorylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. The process of chemical modifications are known in theart, which may include, but are not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the molecules may contain one or more non-classicalamino acids.

The anti-PD-1 antibodies and antigen-binding fragments thereof disclosedherein may include anti-PD-1 antibodies and antigen-binding fragmentsthereof whose binding characteristics have been altered by directmutation, affinity maturation, phage display, or chain shuffling. Theaffinity and specificity may be altered by mutating CDRs and screeningfor CDRs with desired characteristics. Methods of mutagenesis are knownto one of skill in the art.

Antibody Binding

Also provided herein are anti-PD-1 antibodies and antigen-bindingfragments thereof, and fusion proteins comprising anti-PD-1 antibodiesor antigen-binding fragments thereof, that bind to the same epitope onPD-1 as one of the anti-PD-1 antibodies or antigen-binding fragmentsthereof disclosed herein.

In some embodiments, the anti-PD-1 antibodies and antigen-bindingfragments thereof provided herein bind selectively to PD-1 over one ormore PD family members. In one embodiment, the anti-PD-1 antibody orantigen-binding fragment thereof does not exhibit significant binding toother PD family members, including to CTLA-4 and/or CD28. The human PD-1gene produces four alternatively spliced PD-1 mRNA transcripts. One ofthese variants produces a soluble form of PD-1. In one aspect, thedisclosure provides anti-PD-1 antibodies or antigen-binding fragmentsthereof that bind to all isoforms of PD-1. In some embodiments, theprovided anti-PD-1 antibodies and antigen-binding fragments thereof bindto mammalian PD-1.

As used herein, “binding” of an antibody or antigen-binding fragmentthereof, or fusion protein comprising an antibody or antigen-bindingfragment thereof, to PD-1, an epitope on PD-1, or, in certainembodiments described below, particular residues on PD-1, includes theselective interaction of the antibody or antigen-binding fragmentthereof with PD-1. Binding therefore includes, e.g., primary andsecondary interactions including hydrogen bonds, ionic interactions,salt bridges, as well as hydrophilic and hydrophobic interactions.

In certain embodiments, the anti-PD-1 antibodies or antigen-bindingfragments thereof described herein bind to PD-1 with an equilibriumconstant for the dissociation (K_(D)) of 10⁻² to 10⁻¹⁰ mol/l, 10⁻³ to10⁻¹⁰ mol/l, 10⁻⁴ to 10⁻¹⁰ mol/l, 10⁻⁵ to 10⁻¹⁰ mol/l, 10⁻⁶ to 10⁻¹⁰mol/l, 10⁻⁷ to 10⁻¹⁰ mol/l, 10⁻⁸ to 10⁻¹⁰ mol/l or 10⁻⁹ to 10⁻¹⁰ mol/l.In other embodiments, the anti-PD-1 antibodies or antigen-bindingfragments thereof described herein bind to PD-1 with a K_(D) of 10⁻² to10⁻⁹ mol/l, 10⁻³ to 10⁻⁹ mol/l, 10⁻⁴ to 10⁻⁹ mol/l, 10⁻⁵ to 10⁻⁹ mol/l,10⁻⁶ to 10⁻⁹ mol/l, 10⁻⁷ to 10⁻⁹ mol/l, 10⁻⁸ to 10⁻⁹ mol/l, or 10⁻⁹10⁻¹⁰ mol/l.

As used herein, “affinity,” represented by the K_(D) of an antigen withan antigen-binding protein, is a measure of the binding strength betweenan antigenic determinant and an antigen-binding site on theantigen-binding protein, such as an antibody or antibody fragmentthereof. The value of K_(D) is inversely proportional to the bindingstrength between an antigenic determinant and the antigen-bindingmolecule. Alternatively, the affinity can also be expressed as theassociation constant (K_(A)), which is 1/K_(D)). Affinity can bedetermined in a manner known per se, depending on the specific antigenof interest by a person skilled in the art.

The term “specificity” herein refers to the ability of an antibody orantigen-binding fragment thereof, such as an anti-PD-1 antibody orantigen-binding fragment thereof, to recognize an epitope within PD-1,while only having little or no detectable reactivity with other portionsof PD-1. Specificity can be relatively determined by competition assaysor by epitope identification/characterization techniques describedherein or their equivalents known in the art.

The term “epitope” herein refers to the specific target to which anantibody binds. Epitopes can be formed both by a contiguous stretch ofamino acids (continuous epitopes) and by three-dimensional arrangementof amino acid residues that exists only when the target protein isfolded in a particular conformation (discontinuous epitopes.) Ingeneral, an epitope comprises at least 3 amino acids, at least 4, atleast 5, or about 7-10 amino acids.

Disclosed herein are anti-PD-1 antibodies and antigen-binding fragmentsthereof as well as fusion proteins comprising an anti-PD1 antibody orantigen-binding fragment thereof that specifically bind to the sameepitope as anti-PD-1 antibody 38B2. Also disclosed herein are anti-PD-1antibodies and antigen-binding fragments thereof as well as fusionproteins comprising an anti-PD1 antibody or antigen-binding fragmentthereof that bind to the same epitope as anti-PD-1 antibody 31B1.

As used herein, a “blocking” antibody or an antibody “antagonist” is onethat inhibits or reduces the biological activity of the antigen to whichit binds. For example, in some embodiments, an anti-PD-1 antagonistantibody or antigen-binding fragment thereof binds PD-1 and inhibitsactivity of PD-1 and/or binding of PD-1 to binding partners such asPD-L1 or PD-L2. Inhibition of activity and inhibition of bindingincludes partial inhibition. Methods for the identification of PD-1antibodies that block PD-1 interactions are described herein and areknown to the ones skilled in the art. For instance, competing,cross-blocking, and cross-blocked antibodies can be identified using anysuitable method known in the art, including competition ELISAs orBIACORE® assays where binding of the competing or cross-blockingantibody to human PD-1 prevents the binding of an antibody disclosedherein or vice versa.

In certain embodiments, not all CDRs are directly involved in binding tothe antigen. In one embodiment, four out of six CDRs of the anti-PD-1antibody or antigen-binding fragment thereof make contact with theantigen. In one embodiment, five out of six CDRs of the anti-PD-1antibody or antigen-binding fragment thereof make contact with theantigen. In one embodiment, six out of six CDRs of the anti-PD-1antibody or antigen-binding fragment thereof make contact with theantigen.

The terms “selective” and “selectivity” herein refer to the preferentialbinding of an antibody or antigen-binding fragment thereof (i.e., a PD-1antibody or antigen-binding fragment thereof), for a particular region,target, or peptide; typically a region or epitope in PD-1, as opposed toone or more other biological molecules, including other PD-1 familymembers.

In one aspect, provided are anti-PD-1 antibodies and antigen-bindingfragments thereof that specifically bind to at least part of the bindingsite on PD-1, thereby blocking PD-1 interactions with the one or morePD-1 ligands. These PD-1 ligands include, but are not limited to, PD-L1and PD-L2.

In certain embodiments, the anti-PD-1 antibody or antigen-bindingfragments according to the disclosure comprises an Fc domain, composedof a first and a second subunit. The Fc domain of an antibody consistsof a pair of polypeptide chains comprising heavy chain domains of animmunoglobulin molecule. The two subunits of the Fc domain form a stableassociation. In embodiments, the two subunits of the Fc domain areidentical. In alternative embodiments, the two subunits of the Fc domainare non-identical. In embodiments, one subunit of the Fc domain may befused with an immunoconjugate molecule. In embodiments, the Fc domain ofthe antibody may be an IgG Fc domain, an IgG1 Fc domain, an IgG2Fcdomain, an IgG3 Fc domain, an IgG4 Fc domain. In a further particularembodiment, the Fc domain is a human Fc domain.

Fc Domain Modifications Promoting Heterodimerization

Further contemplated are modifications in the Fc domain of the disclosedanti-PD-1 antibodies or antigen-binding fragments thereof promotingdimerization. In embodiments, the Fc domain of the anti-PD-1 antibody orantigen-binding fragment thereof comprises a modification promoting theassociation of the first and the second subunit of the Fc domain. In oneembodiment, said modification is in the CH3 domain of the Fc domain. Ina specific embodiment, said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

Knob-into-hole modifications are a “protuberance-into-cavity” strategy,which serves to engineer an interface between a first and secondpolypeptide for hetero-oligomerization. “Protuberances” (i.e., theknobs) are constructed by replacing small amino acid side chains fromthe interface of the first polypeptide with larger side chains.Compensatory “cavities” (i.e., holes) of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide by replacing large amino acid side chains with smaller ones.In particular embodiment, an amino acid residue in the CH3 domain of thefirst Fc subunit is replaced with an amino acid residue having a largerside chain volume, thereby generating a knob within the CH3 domain ofthe first Fc subunit which is positionable in a hole present within theCH3 domain of the second Fc subunit, generated by replacing one aminoacid residue with an amino acid residue having a smaller side chainvolume in the CH3 domain of the second Fc subunit. Preferably said aminoacid residue having a larger side chain volume is selected from thegroup consisting of cysteine (C), valine (V), alanine (A), phenylalanine(F), tyrosine (Y), leucine (L), lysine (K), Proline (P), glutamic acid(E), and tryptophan (W). Preferably said amino acid residue having asmaller side chain volume is selected from the group consisting ofalanine (A), serine (S), threonine (T), arginine (R), tryptophan (W),cysteine (C), lysine (L), glutamic acid (E), aspartic acid (D), andvaline (V).

The mutations corresponding to the knob and the hole can be made byaltering the nucleic acid encoding the polypeptides, e.g. bysite-specific mutagenesis, or by peptide synthesis.

In fusion protein embodiments, which only one heavy chain is linked toIL-15/IL-15Rα sushi, the Fc domains of the anti-PD-1 antibody orantigen-binding fragment thereof may comprise one or more amino acidsubstitutions promoting heterodimer formation (i.e., the association theheavy chain fusion with a heavy chain lacking the fusion). Inembodiments, an amino acid residue in the CH3 domain of one heavy chain(CH3-1) comprises an amino acid substitution replacing an amino acidwith an amino acid residue having a larger side chain volume, therebygenerating a “knob” within the CH3 domain, which is positionable in a“hole” present within the CH3 domain of the other heavy chain (CH3-2),generated by replacing an amino acid residue with an amino acid residuehaving a smaller side chain volume. In embodiments, the CH3 domain ofthe first subunit of the Fc domain (the “knobs” subunit) may compriseone or more substitution from the following; T350V, L351Y, S354C, S364H,T366Y, T366W, F405A, Y407V. In specific embodiments, the CH3 domain ofthe second subunit of the Fc domain (the “holes” subunit) may compriseone or more substitutions from the following; Y349C, T350V, T366L,T366S, L368A, K392L, T394W, Y407V, Y407T.

In some embodiments, the amino acid substitutions are selected from:

-   -   (1) CH3 domain of first heavy chain: S354C, T366W; CH3 domain of        second heavy chain: Y349C, T366S, L368A, Y407V;    -   (2) CH3 domain of first heavy chain: T350V, L351Y, F405A, Y407V;        CH3 domain of second heavy chain: T350V, T366L, K392L, T394W;    -   (3) CH3 domain of first heavy chain: L351Y, F405A, Y407V; CH3        domain of second heavy chain: T366L, K392L, T394W;    -   (4) CH3 domain of first heavy chain: T366W; CH3 domain of second        heavy chain: Y407T;    -   (5) CH3 domain of first heavy chain: T366Y; CH3 domain of second        heavy chain: Y407T;    -   (6) CH3 domain of first heavy chain: T366W; CH3 domain of second        heavy chain: Y407A;    -   (7) CH3 domain of first heavy chain: F405A; CH3 domain of second        heavy chain: T394W;    -   (8) CH3 domain of first heavy chain: F405W; CH3 domain of second        heavy chain: T394S;    -   (9) CH3 domain of first heavy chain: T366Y and F405A; CH3 domain        of second heavy chain: T394W and Y407T;    -   (10) CH3 domain of first heavy chain: T366W and F405W; CH3        domain of second heavy chain: T394S: and 407A;    -   (11) CH3 domain of first heavy chain: F405W and Y407A; CH3        domain of second heavy chain: T366W and T394S.    -   (12)

Provided herein is an antibody or antigen-binding fragment thereof,which binds to PD-1 (or a fusion protein comprising such an antibody orantigen-binding fragment thereof), wherein the antibody comprises aheavy chain comprising a sequence selected from the group consisting ofSEQ ID NOS: 168-169, 171-172, 185, 191, and 201-203.

Provided herein is an antibody or antigen-binding fragment thereof (or afusion protein comprising such an antibody or antigen-binding fragmentthereof), which binds to PD-1, wherein the antibody comprises a lightchain comprising a sequence selected from the group consisting of SEQ IDNOS: 170, 173, 186, 192, and 205.

In specific embodiments, the Fc domain according to the disclosureexhibits reduced binding affinity to an Fc receptor and/or reducedeffector function, as compared to a native IgG1 Fc domain. In certainembodiments, the Fc domain is engineered to have reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a non-engineered Fc domain.

Linkers

In embodiments, the fusion proteins provided herein may comprise one ormore linkers joining components of the fusion proteins disclosed herein.A linker may be located (i) between the IL-15 polypeptide and the IL15Rαpolypeptide comprising the sushi domain; (ii) between a heavy chain ofthe anti-PD-1 antibody or antigen-binding fragment thereof and the IL-15polypeptide or the IL15Rα polypeptide comprising the sushi domain; or(iii) both. In embodiments of the disclosure, the IL-15 polypeptide andthe IL15Rα polypeptide comprising the sushi domain are joined or linkedby a first linker amino acid sequence. In embodiments, the IL-15polypeptide (or the IL15Rα polypeptide) is linked to an antibody, orantigen binding fragment thereof, described herein by a second linkeramino acid sequence. In embodiments, the second linker joins the Nterminus of the heavy chain variable region to the C terminus of theIL-15 polypeptide. The first and second linkers may have the same ordifferent amino acid sequences.

The linker amino acids sequences described herein may be of a lengthsufficient to ensure that the fusion protein forms proper secondary andtertiary structures. The length of the linker amino may be between 5 to40 amino acids, preferably 10 to 40 amino acids, more preferably 15 to40 amino acids, still more preferably 20 to 40 amino acids, mostpreferably 25 to 35 amino acids.

Preferably, the linker sequences comprise near neutral amino acidsselected in the group comprising Gly (G), Asn (N), Ser (S), Thr (T), Ala(A), Leu (L), and Gln (Q), most preferably in amino acids selected fromthe group comprising Gly (G), Asn (N), and Ser (S). Preferably, thelinker sequences are glycine- and serine-rich, and in some embodiments,the linker contains only serine and glycine residues.

In some embodiments, the linker comprises a portion of the IL15Rαpolypeptide outside of the sushi domain, including, but not limited tothe sequence bolded in the linker of SEQ ID NO:215 (below).

Some non-limiting linker examples of amino acid sequences include

(SEQ ID NO: 215) IRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQ, (SEQ ID NO: 216)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; (SEQ ID NO: 217)IRDPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG, (SEQ ID NO: 218)IRDPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS, and (SEQ ID NO: 244)GGGGSGGGGSGGGGSGGGGS.

In embodiments, the linker joining the IL-15 polypeptide and the IL-15Rαsushi comprises IRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 215),IRDPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG (SEQ ID NO: 217), orIRDPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:218).

In embodiments, the linker joining the IL-15 polypeptide to theanti-PD-1 antibody heavy chain comprises GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 216) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:244).

In embodiments, the heavy and light chains of the anti-PD-1 antibody orantigen-binding fragment thereof disclosed herein may be connected intoa single polypeptide chain (a “single-chain Fv” or “scFv”) using a thirdlinker that allows the VR and VL domains to associate to form anantigen-binding site. The amino acid sequence of the linkers may be thesame or different.

In one embodiment, an IL-15 polypeptide or an IL-15 derivative iscovalently linked to an IL-15Rα sushi polypeptide or an IL-15Rα sushiderivative by a linker.

In one embodiment, the IL-15 polypeptide or IL-15 derivative iscovalently linked to an anti-PD-1 antibody or antigen-binding fragmentthereof by a linker. In one embodiment, the C-terminus of the IL-15polypeptide or IL-15 derivative is covalently linked to the N-terminusof the anti-PD-1 antibody or antigen-binding fragment thereof by alinker. In one embodiment, the N-terminus of the IL-15 polypeptide orIL-15 derivative is covalently linked to the C-terminus of the anti-PD-1antibody or antigen-binding fragment thereof by a linker. In oneembodiment, the N-terminus of the IL-15 polypeptide or IL-15 derivativeis covalently linked to the CH3 region of the anti-PD-1 antibody orantigen-binding fragment thereof by a linker.

In one embodiment, the IL-15Rα sushi or an IL-15Rα sushi derivative iscovalently linked to an anti-PD-1 antibody or antigen-binding fragmentthereof by a linker. In one embodiment, the C-terminus of the IL-15Rαsushi polypeptide or IL-15Rα sushi derivative is covalently linked tothe N-terminus of the anti-PD-1 antibody or antigen-binding fragmentthereof by a linker. In one embodiment, the N-terminus of the IL-15Rαsushi polypeptide or IL-15Rα sushi derivative is covalently linked tothe C-terminus of the anti-PD-1 antibody or antigen-binding fragmentthereof by a linker. In one embodiment, the N-terminus of the IL-15Rαsushi polypeptide or IL-15Rα sushi derivative is covalently linked tothe CH3 region of the anti-PD-1 antibody or antigen-binding fragmentthereof by a linker.

In some embodiments, an IL-15 polypeptide or an IL-15 derivative iscovalently linked to an IL-15Rα sushi polypeptide or a IL-15Rα sushiderivative by a first linker and either the IL-15 polypeptide or IL-15derivative or the IL-15Rα sushi polypeptide or IL-15Rα sushi derivativeis covalently linked to an anti-PD-1 antibody or antigen-bindingfragment thereof by a second linker. In some embodiments, the amino acidsequences of the first and the second linker are identical. In otherembodiments, the amino acid sequences of the first and the second linkerare different.

Conjugates

The anti-PD-1 antibodies, antigen-binding fragments thereof, and thefusion proteins disclosed herein may further comprise one or morefunctional moieties. Examples of useful functional moieties include, butare not limited to, a blocking moiety, a detectable moiety, a diagnosticmoiety, a targeting, and a therapeutic moiety.

A blocking moiety may include moieties of sufficient steric bulk and/orcharge such that reduced glycosylation occurs, for example, by blockingthe ability of a glycosidase to glycosylate the antibody orantigen-binding fragment thereof. Preferred blocking moieties includecysteine adducts such as cysteine, mixed disulfide adducts, or disulfidelinkages and PEG moieties such as polyethylene glycol (“PEG”),polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”) andother polyoxyethylated polyols, polyvinyl alcohol (“PVA”) and otherpolyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylatedglucose. PEG is a preferred moiety in biological applications forseveral reasons. PEGylating can improve pharmacokinetic performance of amolecule by increasing the molecule's apparent molecular weight. Theincreased apparent molecular weight reduces the rate of clearance fromthe body following subcutaneous or systemic administration. In manycases, pegylation can decrease antigenicity and immunogenicity.PEGylating can also increase the solubility of a biologically activemolecule. Additionally, PEG typically is clear, colorless, odorless,soluble in water, stable to heat, inert to many chemical agents, doesnot hydrolyze, and is nontoxic, making it a preferable choice forbiological applications.

The examples of detectable moieties that can be conjugated with theanti-PD-1 antibody or the antigen-binding fragments or fusions disclosedherein may include fluorescent moieties or labels, imaging agents,radioisotopic moieties, radiopaque moieties, and the like, e.g.detectable labels such as biotin, fluorophores, chromophores, spinresonance probes, or radiolabels. Examples of fluorophores includefluorescent dyes (e.g. fluorescein, rhodamine, and the like) and otherluminescent molecules (e.g. luminal). A fluorophore may beenvironmentally-sensitive such that its fluorescence changes if it islocated close to one or more residues in the modified protein thatundergo structural changes upon binding a substrate (e.g. dansylprobes). Exemplary radiolabels include small molecules containing atomswith one or more low sensitivity nuclei (13C, 15N, 2H, 125I, 123I, 99Tc,43K, 52Fe, 67Ga, 68Ga, 111In and the like).

Diagnostic moieties include detectable moieties suitable for revealingthe presence of a disease or disorder. Typically, a diagnostic moietyallows for determining the presence, absence, or levels of a molecule,for example, a target peptide, protein, or proteins, that are associatedwith a disease or disorder. Such diagnostics are also suitable forprognosing and/or diagnosing a disease or disorder and its progression.

Examples of therapeutic moieties include anti-inflammatory agents,anti-cancer agents, anti-neurodegenerative agents, anti-infectiveagents, or generally a therapeutic. The functional moiety may also haveone or more of the above-mentioned functions. Exemplary therapeuticmoieties may include an antibiotic, a second anti-PD-1 antibody, or anantibody to another antigen such a tumor-specific antigen, an autoimmunetissue antigen, a virally-infected cell antigen, a Fc receptor, a T cellreceptor, or a T cell co-inhibitor, or an immunotoxin, or any othertherapeutic moiety useful for treating a disease or condition includingcancer, autoimmune disease or chronic viral infection. Exemplarytherapeutic moieties may also cytotoxin, radioactive agent, cytokine,interferon, target or reporter moiety, enzyme, toxin, peptide ortherapeutic agent at any location along the molecule so long as it isable to bind its target. Examples of immunoconjugates include antibodydrug conjugates and antibody-toxin fusion proteins. In certainembodiments, the antibody may be conjugated to an agent specific for atumor cell or a virally infected cell.

A salvage receptor binding epitope as described, e.g., in U.S. Pat. No.5,739,277 may also be attached to the antibody or antigen-bindingfragment thereof (especially an antibody fragment) to increase thehalf-life of the antibodies or the antigen-binding fragments describedherein. The term “salvage receptor binding epitope” may refer to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule (e.g., Ghetie et al., 18 Ann. Rev. Immunol. 739 (2000).

Nucleic Acids

Also provided herein are nucleic acids encoding anti-PD-1 antibodies,antigen-binding fragments thereof and fusion proteins disclosed herein,as well as vectors, host cells, and expression systems. The term“nucleic acid” as used herein refers to a polymeric form of nucleotidesof any length, either ribonucleotides or desoxyribonucleotides andincludes but is not limited to, single-, double- or multi-stranded DNAor RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprisingpurine and pyrimidine bases, or other natural, chemically orbiochemically modified, non-natural, or derivatized nucleotide bases.The nucleic acids encoding anti-PD-1 antibodies and antigen-bindingfragments thereof and fusion proteins disclosed herein may be, e.g.,DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantlyproduced chimeric nucleic acid molecule comprising any of thosepolynucleotides either alone or in combination.

The term “vector” refers to vehicle comprising a nucleic acid moleculethat is capable of transporting the nucleic acid molecule into a cell. A“vector” includes, but is not limited to, a viral vector, a plasmid, aRNA vector or a linear or circular DNA or RNA molecule, which mayconsists of a chromosomal, non-chromosomal, semi-synthetic or syntheticnucleic acids. In some embodiments, the employed vectors are thosecapable of autonomous replication (episomal vector) and/or expression ofnucleic acids to which they are linked (expression vectors). A number ofsuitable vectors are known to those of skill in the art and arecommercially available.

Antibody and Fusion Protein Preparation and Expression Systems

The anti-PD-1 antibodies, antigen-binding fragments or the fusionproteins disclosed herein are typically produced by recombinantexpression. Nucleic acids encoding light and heavy chain variableregions, optionally linked to constant regions, may be inserted into thesame expression vectors. Alternatively, the nucleic acids encoding lightand heavy chain variable regions, optionally linked to constant regions,are inserted into different expression vectors. The expression vectormay further comprise one or more expression control sequences, whichinclude, but are not limited to, promoters (e.g., homologous orheterologous promoters), signal sequences, enhancer elements, andtranscription termination sequences. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Typically, the hostis maintained under conditions suitable for high-level expression of thenucleotide sequences, and the collection and purification of thecross-reacting antibodies after the vector is incorporated into theappropriate host.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences.

The host used to express the anti-PD-1 antibodies, antigen-bindingfragments thereof or the fusion proteins disclosed herein can be aprokaryotic or eukaryotic host. Examples of suitable hosts includebacterial or eukaryotic hosts, including yeast, insects, fungi, bird andmammalian cells either in vivo, or in situ, or host cells of mammalian,insect, bird, or yeast origin. The mammalian cell or tissue can be ofhuman, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat,dog, or cat origin, but any other mammalian cell may be used.

Examples of bacterial hosts that can be used to express the antibodies,antigen-binding fragments or the fusion protein disclosed herein can beE. coli, bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species.

Yeasts may also be used as hosts for expressing the express theantibodies, antigen-binding fragments or the fusion protein disclosedherein. Saccharomyces and Pichia are exemplary yeast hosts, withsuitable vectors having expression control sequences (e.g., promoters),an origin of replication, termination sequences, and the like asdesired. Typical promoters include 3-phosphoglycerate kinase and otherglycolytic enzymes. Inducible yeast promoters include, among others,promoters from alcohol dehydrogenase, isocytochrome C, and enzymesresponsible for methanol, maltose, and galactose utilization.

Mammalian cells in culture may also be used as host cells for expressingthe antibodies, antigen-binding fragments or the fusion proteinsdisclosed herein. Examples of suitable host cell lines capable ofsecreting heterologous proteins (e.g., intact immunoglobulins) which arewell known in the art, include CHO cell lines, various COS cell lines,HeLa cells, 293 cells, myeloma cell lines, transformed B-cells, andhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer and necessary processing information sites such as ribosomebinding site, RNA splice site and/or transcriptional terminatorsequences. Examples of expression control sequences include SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like.

The anti-PD-1 antibodies, antigen-binding fragments thereof, and thefusion proteins disclosed herein can be expressed using a singleexpression construct or vector or multiple expression constructs orvectors (e.g., two or three expression constructs). When the antibodyheavy and light chains are cloned on separate expression vectors, thevectors are co-transfected to obtain expression and assembly of intactimmunoglobulins. Once expressed, the whole antibodies, their dimers,individual light and heavy chains, or other immunoglobulin formsdisclosed herein can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, HPLC purification, gel electrophoresis, and the like(see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,(1982)). Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses.

The disclosed anti-PD-1 antibodies, antigen-binding fragments, and thefusion proteins can be made by any method known in the art. Generaltechniques for generating human or mouse antibodies or fusion moleculesare known in the art.

Methods for Modulating PD-1 Activity

In one aspect, the disclosure provides methods of using the anti-PD-1antibodies, antigen-binding fragments thereof, and fusion proteinsdescribed herein for decreasing the interaction between PD-1 andPD-ligands, including, but not limited to, PD-L1 and PD-L2. In someembodiments, the anti-PD-1 antibody, antigen-binding fragment thereof,or fusion protein disrupts the interaction between PD-1 monomers.

The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusionproteins disclosed herein are useful for reducing immunosuppression,e.g., T cell tolerance. By “reducing” is meant the ability to cause anoverall decrease of about 20% or greater, 30% or greater, 40% orgreater, 45% or greater, 50% or greater, of 55% or greater, of 60% orgreater, of 65% or greater, of 70% or greater, or 75% or greater, 80% orgreater, 85% or greater, 90% or greater, or 95% or greater, as comparedto a control that is not treated. Immunosuppression can be mediated byimmune inhibitory receptors expressed on the surface of an immune cell,and their interactions with their ligands. Methods of measuring T cellactivity are known in the art. By way of non-limiting example, T celltolerance can be induced by contacting T cells with recall antigen,anti-CD3 in the absence of co-stimulation, and/or ionomycin. Levels of,e.g., IL-27, LDH-A, RAB10, and/or ZAP70 (both intracellular or secreted)can be monitored, for example, to determine the extent of T celltolerogenesis (with levels of IL-2, interferon-7 and TNF correlatingwith increased T cell tolerance).

The anti-PD-1 antibodies, antigen-binding fragments thereof, and fusionproteins disclosed herein are further useful for enhancing T cellexpansion, activation, and proliferation.

Methods of Treatment

PD-1 μlays an important role in the immune system in regulatingautoimmunity, tumor immunity, and infectious immunity. PD-1-mediated Tcell inhibition is an important mechanism to prevent autoimmunity. Inaddition, cancer and chronic infectious diseases may usurp thisregulation mechanism to drive immune suppression. Blocking PD-1 withantagonists, including monoclonal antibodies, has been studied intreatments of cancer and chronic viral infections. The ability of PD-1to inhibit T cell activation is exploited by chronic viral infectionsand tumors to evade immune response. In chronic viral infections, PD-1is highly expressed on virus-specific T cells and these T cells become“exhausted” with loss of effector functions and proliferative capacity.

PD-1 deficient animal models develop various autoimmune phenotypesincluding autoimmune cardiomayopathy, rheumatoid arthritis, and graftversus host disease. Specifically, they have elevated levels of IgG2band IgA and develop mild lupus-like autoimmunity and dilatedcardiomyopathy. Considering the role of PD-1 in modulating immuneresponses, the therapeutic agents disclosed herein that antagonize PD-1signaling can be administered to treat diseases that involvePD-1-mediated immune suppression.

Immunomodulation is a useful therapeutic approach for treating variousdiseases and disorders. One approach to immunomodulation is to interveneat one or more immune checkpoints by regulators of immune activationthat play a key role in maintaining immune homeostasis and preventingautoimmunity. Depending on the disease or disorder, it may be desirableto upregulate or downregulate the immune response. Tumor cells thatdisplay non-self-antigens, can evade immune attack by secretingcytokines or ligands that activate immune checkpoints. In cancertherapy, thus it is generally desirable to upregulate the immuneresponse against tumor cells. In contrast, in treatment of autoimmunediseases, it is generally desirable to downregulate the immune responsein certain tissues.

In one aspect, the disclosure provides anti-PD-1 antibodies,antigen-binding fragments thereof, and fusion proteins that are usefulfor treatments of subjects in need thereof.

In the methods described herein, a therapeutically effective amount ofan anti-PD-1 antibody, antigen-binding fragment thereof or fusionprotein disclosed herein is administered to a mammal in need thereof.The term “mammal” as used herein includes, but is not limited to,humans, laboratory animals, domestic pets, and farm animals. Preferably,the mammal is a human. “Therapeutically effective amount” as describedherein refers to an amount of an anti-PD-1 antibody, antigen-bindingfragment thereof or fusion protein which, when administered to a mammal,is effective in producing the desired therapeutic effect.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline,etc. Individuals and patients are also subjects herein.

The terms “treat,” “treated,” “treating,” or “treatment” as used hereinrefer to therapeutic treatment, wherein the object is to slow down(lessen) an undesired physiological condition, disorder, or disease, orto obtain beneficial or desired clinical results. For the purposes ofthis disclosure, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms; diminishment of the extent ofthe condition, disorder or disease; stabilization (i.e., not worsening)of the state of the condition, disorder or disease; delay in onset orslowing of the progression of the condition, disorder or disease;amelioration of one or more symptoms of the condition, disorder ordisease state; and remission (whether partial or total). Treatmentincludes eliciting a clinically significant response without excessivelevels of side effects. Treatment also includes prolonging survival ascompared to expected survival if not receiving treatment. The terms“prevent,” “prevention,” and the like refer to acting prior to overtdisease or disorder onset, to prevent the disease or disorder fromdeveloping or to minimize the extent of the disease or disorder or slowits course of development.

In one aspect, the anti-PD-1 antibodies, antigen-binding fragmentsthereof and fusion proteins disclosed herein may be used to treatsubjects suffering from an autoimmune disease, including but not limitedto, alopecia areata, autoimmune hepatitis, celiac disease, Graves'disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia,inflammatory bowel disease, inflammatory myopathies, multiple sclerosis,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma,Sjogren's syndrome, systemic lupus, erthyematosus, vitiligo, autoimmunepancreatitis, autoimmune urticaria, autoimmune thrombocytopenic purpura,Crohn's disease, diabetes type I, eosinophilic fasciitis, eosinophilicenterogastritis, Goodpasture's syndrome, myasthenia gravis, psoriaticarthritis, rheumatic fever, ulcerative colitis, vasculitis and Wegener'sgranulomatosis. In certain embodiments, an activating antibody disclosedherein may be used to treat a subject suffering from autoimmune disease.

In one aspect, the anti-PD-1 antibodies, antigen-binding fragmentsthereof and fusion proteins disclosed herein may be used to treatsubjects suffering from a chronic viral infection. In a specificembodiment, the anti-PD-1 antibodies, antigen-binding fragments thereofand fusion proteins disclosed herein can be used to rescue exhausted Tcells and/or decrease viral titers in a subject in need thereof. Incertain embodiments, the anti-PD-1 antibodies, antigen-binding fragmentsthereof and fusion proteins disclosed herein may be used to treatchronic viral infection by administering a therapeutically effectivedose to a subject in need thereof. In some embodiments, the subject issuffering from a viral infection caused by lymphocytic choriomeningitisvirus (LCMV), human immunodeficiency virus (HIV) or human papillomavirus (HPV) or hepatitis B/C virus (HBV/HCV) or simian immunodeficiencyvirus (SIV).

In another aspect, the anti-PD-1 antibodies, antigen-binding fragmentsthereof and fusion proteins disclosed herein can be used to treatsubjects suffering from primary or recurrent cancer, including, but notlimited to, renal cell carcinoma, colorectal cancer, non-small-cell lungcancer, brain cancer (e.g., glioblastoma multiforme), squamous cellcarcinoma of head and neck, gastric cancer, prostate cancer, ovariancancer, kidney cancer, breast cancer, multiple myeloma, and melanoma.

In certain embodiments, anti-PD-1 antibodies, antigen-binding fragmentsthereof and fusion proteins disclosed herein may be administered in atherapeutically effective amount to a subject suffering from a cancer ora viral infection.

One or more anti-PD-1 antibodies, antigen-binding fragments thereof orfusion proteins disclosed herein may be administered to relieve orprevent or decrease the severity of one or more of the symptoms orconditions of the disease or disorder. In a certain embodiment, theanti-PD-1 antibodies, antigen-binding fragments thereof and fusionproteins disclosed herein may be administered prophylactically to asubject in need therefore to reduce the risk of developing a chronicviral infection or an autoimmune disease. The anti-PD-1 antibodies,antigen-binding fragments thereof and fusion proteins disclosed hereinmay be used as an adjunct therapy with any other agent or any othertherapy known to those skilled in the art useful for treating cancer,autoimmune disease, or viral infection.

Combination Therapy

The anti-PD-1 antibodies, antigen-binding fragments thereof and fusionproteins disclosed herein may be advantageously combined with anadditional therapeutic agent. Such additional agents include, but arenot limited to, cytotoxic agents, chemotherapeutic agents, growthinhibitory agents, anti-inflammatory agents, anti-cancer agents,anti-neurodegenerative agents, immunosuppressive agents, andanti-infective agents. The administration of the anti-PD-1 antibody orantigen-binding fragment thereof or the fusion protein and theadditional therapeutic agent may be concurrently, consecutively orintermittently. The administration of the anti-PD-1 antibody orantigen-binding fragment thereof or the fusion protein and theadditional therapeutic agent may be separately or as a mixture. Further,the methods of treatment provided herein can relate to a treatment incombination with one or more therapies including but not limited to thegroup of antibody therapy, chemotherapy, cytokine therapy, dendriticcell therapy, gene therapy, hormone therapy, laser light therapy, andradiation therapy.

The anti-PD-1 antibodies, antigen-binding fragments thereof, and thefusion proteins of the present disclosure may be combinedsynergistically with one or more anti-cancer drugs or therapy used totreat cancer, including, but not limited to, renal cell carcinoma,colorectal cancer, glioblastoma multiforme, squamous cell carcinoma ofhead and neck, non-small-cell lung cancer, colon cancer, ovarian cancer,adenocarcinoma, prostate cancer, glioma, and melanoma. Examples of suchagents include but are not limited to with an antibody to PD-L1, asecond antibody to PD-1 (e.g., nivolumab), a LAG-3 inhibitor, a CTLA-4inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, an antagonist of another T cellco-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAI R 1,ICOS, CD1 60 or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor,a vascular endothelial growth factor (VEGF) antagonist [e.g., a“VEGF-Trap”such as aflibercept or other VEGF-inhibiting fusion proteinas set forth in U.S. Pat. No. 7,087,411, or an anti-VEGF antibody orantigen-binding fragment thereof (e.g., bevacizumab, or ranibizumab) ora small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib,sorafenib, or pazopanib)], an Ang2 inhibitor (e.g., nesvacumab), atransforming growth factor β (TGF3) inhibitor, an epidermal growthfactor receptor (EGFR) inhibitor (e.g., erlotinib, cetuximab), anagonist to a costimulatory receptor (e.g., an agonist toglucocorticoid-induced TNFR-related protein), an antibody to atumor-specific antigen (e.g., CA9, CA1 25, melanoma-associated antigen 3(MAGE3), carcinoembryonic antigen (CEA), anti-viral drugs (e.g.,zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz,cobicistat, tenofovir, rilpivirine and corticosteroids), vimentin,tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9), a vaccine (e.g., Bacillus Calmette-Guerin, a cancer vaccine), anadjuvant to increase antigen presentation (e.g., granulocyte-macrophagecolony stimulating factor), a bispecific antibody (e.g., CD3×CD20bispecific antibody, PSMA×CD3 bispecific antibody), cancer vaccines(e.g., MAGE3, MUC1, EGFRv3, ALVAC-CEA), a cytotoxin, a chemotherapeuticagent (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel,doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine,methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine),cyclophosphamide, radiotherapy, an IL-6R inhibitor (e.g., sarilumab), anIL-4R inhibitor (e.g., dupilumab), an IL-1 0 inhibitor, a cytokine suchas IL-2, IL-7, IL-21, and IL-15, an antibody-drug conjugate (ADC) (e.g.,anti-CD1 9-DM4 ADC, and anti-DS6-DM4 ADC), an anti-inflammatory drug(e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), adietary supplement such as anti-oxidants or any palliative care to treatcancer, radiation therapy, and/or an antibody to a Fc receptor on immunecells for the treatment of an autoimmune disease.

Methods of Administration

The therapeutic compositions comprising any of the anti-PD-1 antibodies,antigen-binding fragments thereof or fusion proteins described hereinmay be administered to a subject in need thereof in any convenientmanner including but not limited to by injection, transfusion,implantation or transplantation. The compositions described herein maybe administered to a subject in need thereof subcutaneously,intradermally, intratumorally, intranodally, intramedullary,intramuscularly, intracranially, by intravenous or intralymphaticinjection, or intraperitoneally. In one embodiment, the cellcompositions of the present disclosure are preferably administered byintravenous injection.

In certain embodiments, the anti-PD-1 antibody, antigen-binding fragmentthereof or the fusion protein is administered to the mammal byintravenous infusion, i.e., introduction of the anti-PD-1 antibody,antigen-binding fragment thereof or the fusion protein into the vein ofa mammal over a certain period. In certain embodiments, the period isabout 5 min, about 10 min, about 30 min, about 1 h, about 2 h, about 4h, or about 8 h.

Administrative Regimens

The methods according to this aspect of the disclosure comprisesequentially administering to a subject multiple doses of an anti-PD-1antibody, antigen-binding fragment thereof, or a fusion protein of thedisclosure. As used herein, “sequentially administering,” means thateach dose of an anti-PD-1 antibody, antigen-binding fragment thereof, ora fusion protein of the disclosure is administered to the subject at adifferent point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentdisclosure includes methods, which comprise sequentially administeringto the patient a single initial dose of an anti-PD-1 antibody,antigen-binding fragment thereof, or a fusion protein of the disclosure,followed by one or more secondary doses of an anti-PD-1 antibody,antigen-binding fragment thereof, or a fusion protein of the disclosure,and optionally followed by one or more tertiary doses of an anti-PD-1antibody, antigen-binding fragment thereof, or a fusion protein of thedisclosure. An anti-PD-1 antibody, antigen-binding fragment thereof, ora fusion protein of the disclosure may be administered at a dose between0.01 mg/kg to 100 mg/kg.

In certain embodiments, a dose of a compound or a composition isadministered to a subject every day, every other day, every couple ofdays, every third day, once a week, twice a week, three times a week,once every two weeks, or once a month. In other embodiments, two, threeor four doses of a compound or a composition is administered to asubject every day, every couple of days, every third day, once a week,once every two weeks or once a month. In some embodiments, a dose(s) ofa compound or a composition is administered for 2 days, 3 days, 5 days,7 days, 14 days, 21 days, or 28 days. In certain embodiments, a dose ofa compound or a composition is administered for 1 month, 1.5 months, 2months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.

Pharmaceutical Compositions

In another aspect, provided are pharmaceutically acceptable compositionsthat comprise a therapeutically effective amount of an anti-PD-1antibody, antigen-binding fragment thereof, or a fusion protein of thedisclosure formulated together with one or more pharmaceuticallyacceptable excipients.

The dosage of active agent(s) may vary, depending on the reason for use,the individual subject, and the mode of administration. The dosage maybe adjusted based on the subject's weight, the age, and health of thesubject, and tolerance for the compound(s) or composition.

The active agent and excipient(s) may be formulated into compositionsand dosage forms according to methods known in the art. Thepharmaceutical compositions provided herein may be specially formulatedin solid or liquid form, including those adapted for parenteraladministration, for example, by subcutaneous, intratumoral,intramuscular or intravenous injection as, for example, a sterilesolution, or suspension.

Therapeutic compositions comprising anti-PD-1 antibodies orantigen-binding fragments thereof, or fusion proteins thereof mayformulated with one or more pharmaceutically-acceptable excipients,which can be a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, carrier,manufacturing aid (e.g., lubricant, talc magnesium, calcium or zincstearate, or steric acid), solvent or encapsulating material, involvedin carrying or transporting the therapeutic compound for administrationto the subject, bulking agent, salt, surfactant and/or a preservative.Some examples of materials which can serve aspharmaceutically-acceptable excipients include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as ethylene glycol and propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents; water; isotonic saline; pH buffered solutions; and othernon-toxic compatible substances employed in pharmaceutical formulations.

A bulking agent as referred herein may be described as a compound addedto increase the mass of a pharmaceutical composition and to contributeto the physical structure of the formulation in the lyophilized form.Examples of bulking agent may include but is not limited to Suitablemannitol, glycine, polyethylene glycol, and sorbitol.

The therapeutic composition may optionally include a surfactant. The useof a surfactant can reduce aggregation of the reconstituted proteinand/or reduce the formation of particulates in the reconstitutedformulation. Examples of suitable surfactants that might be usedaccording to the present disclosure includes but is not limited topolysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, andcopolymers of ethylene and propylene glycol (e.g. Pluronics, PF68,etc.).

A preservative may optionally be used in the therapeutic compositiondescribed herein. Suitable preservatives for use in the formulationprovided herein include octadecyldimethylbenzyl ammonium chloride,hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyl-dimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl, andbenzyl alcohol, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

The therapeutic composition described herein may have a varyingconcentration of the anti-PD-1 antibody, antigen-binding fragmentthereof, or fusion protein. For example, the compositions may comprisean anti-PD-1 antibody, antigen-binding fragment thereof or fusionprotein at 10 mg/ml to 200 mg/ml, 25 mg/ml to 130 mg/ml, 50 mg/ml to 125mg/ml, 75 mg/ml to 110 mg/ml, or 80 mg/ml to 100 mg/ml. The compositionsalso may comprise an anti-PD-1 antibody, antigen-binding fragmentthereof or fusion protein at about 10 mg/ml, 20 mg/ml, 30 mg/ml, 40mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 110mg/ml, 120 mg/ml, 130 mg/ml, 140 mg/ml, or 150 mg/ml. In someembodiments, the therapeutic composition may be lyophilized and providedin a composition for reconstitution prior to administration.

Diagnostic Uses

The anti-PD-1 antibodies, antigen-binding fragments thereof or thefusion proteins of the present disclosure may be used to detect and/ormeasure PD-1 in a sample, e.g., for diagnostic purposes. The anti-PD-1antibodies, antigen-binding fragments thereof or the fusion proteinsdisclosed herein may be used in an assay to detect a disease or disordersuch as cancer, autoimmune disease, or chronic viral infection.Exemplary diagnostic assays for PD-1 may comprise, e.g., contacting asample, obtained from a patient, with an anti-PD-1 antibody,antigen-binding fragment thereof, or a fusion protein of the disclosure,wherein the anti-PD-1 antibody, antigen-binding fragment thereof, or thefusion protein is labeled with a detectable label or reporter moleculeor used as a capture ligand to selectively isolate PD-1 from patientsamples or alternatively used in combination with a secondary antibodywhich is itself detectably labeled. The detectable label or reportermolecule can be a radioisotope, such as 3H, C, 32P, 35S, or 25 1; afluorescent or chemiluminescent moiety such as fluoresceinisothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase,β-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure PD-1 in a sampleinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Kits of the present disclosure can include any combination of agents,compositions, components, reagents, administration devices, ormechanisms, or other entities provided herein. For instance, a kit ofthe present disclosure may include one or more anti-PD-1 antibodies orantigen-binding fragments thereof or fusion proteins disclosed hereinand one or more of a carrier composition, an administration device, anda combination therapy agent. Kits may further include a device tofacilitate delivery such as syringe for injection or a tool thatfacilitates the delivery of therapeutic compositions to the subject inneed thereof. Any of the kits provided herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

All other referenced patents and applications are incorporated herein byreference in their entirety. Furthermore, where a definition or use of aterm in a reference, which is incorporated by reference herein, isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

It is to be understood that this disclosure is not limited to theparticular molecules, compositions, methodologies, or protocolsdescribed, as these may vary. Any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of embodiments of the present disclosure. It is further to beunderstood that the current disclosure in this specification includesall possible combinations of such particular features. For example,where a particular feature is disclosed in the context of a particularaspect or embodiment of the disclosure herein, or a particular claim,that feature can also be used, to the extent possible, in combinationwith and/or in the context of other particular aspects and embodimentsof the disclosure, and in the disclosure generally.

Where reference is made herein to a method comprising two or moredefined steps, the defined steps can be carried out in the order aslisted, or in any order or simultaneously (except where the contextexcludes that possibility), and the method can include one or more othersteps which are carried out before any of the defined steps, between twoof the defined steps, or after all the defined steps (except where thecontext excludes those possibilities).

To facilitate a better understanding of the present disclosure, thefollowing examples of specific embodiments are given. The followingexamples should not be read to limit or define the entire scope of thedisclosure.

EXAMPLES Example 1: Identification of Anti-PD-1 Antibodies from aDistributed Bio SuperHuman 2.0 Inc. Library

hPD-1-Fc (human IgV domain fused to the N-terminal of human IgG1Fc;present as dimer in solution) or hPD-1-his (human IgV domain of PD-1connected to the N-terminal of a six histidine peptide; present asmonomer in solution) was immobilized on immuno-tubes, which were coatedwith an anti-his antibody or an anti-hFc antibody. Biotin-labeledhPD-1-his was captured by strepavidin-conjugated magnetic beads. Thephage library was blocked with 3% PBS milk, added to the antigen-coatedtubes or mixed with antigen-coated beads, and incubated at roomtemperature for 0.5-1 h to allow the phages to bind to the immobilizedantigens. Unbound phages were washed out, while bound phages were elutedwith triethlamine (pH>11). The neutralized phages were used to infectlog phase E. coli, which were grown at 30° C. overnight. The harvestedbacteria were used to amplify the phage for the next round panning.

The heavy and light chain variable domains of selected scFv antibodiesisolated from the phage library were amplified by PCR and inserted intoa mammalian expression vector containing modified human IgG 1 constantdomains, from which the effector functions have been removed. The fulllength IgG antibodies were expressed in HEK293 cells and purified byProtein A. Purified antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11(see SEQ ID NOs: 1-48, Table 25) were characterized by binding (solubleand cell expressed PD-1) and blocking assays, Biacore kinetics analysisand cytokine secretion in SEB stimulated human PBMC.

To identify higher affinity antibodies, two light chain-shufflinglibraries were constructed by combining the heavy chain of lead antibody23H9 or 24H9, respectively, with the light chain mixture from R1panning. Higher-affinity and/or more stable variants were identified bypanning in conditions that were more restricted. These maturedantibodies were converted to IgG, expressed, purified, and characterizedagain. The higher affinity clones were expressed in CHO cells, purifiedby protein A and SE-HPLC to obtain higher quality to run furtherstudies. Antibodies 31B1, 33C4, 33G8, 34C1 (see SEQ ID NOs: 49-65, Table25) were obtained by light chain shuffling using the heavy chain of23H9. Antibodies 32A11, 32D11, 32D2, 32G6, 38A10, 38A11, 38A4, 38A6,38A8, 38B1, 38B10, 38B2, 38C11, 38C6, 38G11, 38G9, 38H3, 39A3, 39B11,39B3, 39B6, 39F11, 39G5, 39G8, 39H11, 39H2, and 39H7 (see SEQ ID NOs:66-138, Table 25) were obtained by light chain shuffling using the heavychain of 24H9.

FIG. 1 represents the sequence alignments of exemplary antibodiesgenerated herein. Table 25 sets forth the amino acid sequenceidentifiers of the heavy and light chain variable regions and CDRs ofselected anti-PD-1 antibodies according to the Kabat CDR definition. Thebiological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2: Antibodies Isolated from Phage Display Library Bind to hPD-1

The ability of anti-hPD1 antibodies 19B6, 19B10, 23A8, 23H9, 24H9, 23A11(isolated from HEK293 cells) to bind to hPD1 was evaluated using a doseresponse binding ELISA using the indicated antibodies as primaryantibodies. For this, high-binding (Immulon 2HB) plates were directlycoated with human PD-1-Fc (R&D, cat #1086-PD-050). Biotin labeledhPD-1-his (Sino Biologicals, cat #10377-H08H) were captured bystrepavidin (Jackson 016-000-114) or anti-his antibody (R&D, cat#MAB050-100) which were coated on the plate one day before. Serialdilutions of antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 wereadded to antigen-coated plates and incubated at room temperature for 1-2hs. After washing the plates with PBST (0.05%) for three times,1/5000-1/10,000 diluted anti-hFab-HRP antibody (Jackson 109-036-097) wasadded to the plates, which were incubated at room temperature for about1 h. Color was developed by adding 1:1 dilution of substrate A and Bfrom TMB (Fisher, cat #5120-0050) followed by neutralizing with 1NH₂SO₄. OD₄₅₀ was read using a TECAN plate reader (Table 1). Themeans±S.D. of the OD₄₅₀ reading was plotted against the log antibodyconcentration using GraphPad Prism8. The EC₅₀ value was calculated byplotting log (agonist) vs. response (three parameters) or log(agonist)vs. response-variable slope (four parameters) (FIG. 2 ). The data pointsin FIG. 2 represent the mean±S.D. of duplicate determinations. All sixantibodies exhibited similar EC₅₀ and OD₄₅₀ (10 nM) values when bound tohuman PD-1-Fc, but exhibited different EC₅₀ and OD₄₅₀ (10 nM) valueswhen bound to PD-1-his (Table 1). Antibodies 23A8, 23H9, and 24H9 showedsuperior binding to hPD-1-his compared to the other antibodies (FIG. 2). Three lead antibodies—23A8, 23H9 and 24H9—were selected based ontheir binding to PD-1-his (Table 1 and FIG. 2 ).

TABLE 1 EC50 and OD at 10 nM values for binding of antibodies 19B6,19B10, 23A8, 23H9, 24H9, and 23A11 to PD-1-Fc or PD-1-his. 19B6 19B1023A8 23H9 24H9 23A11 PD-1- EC50 [nM]   0.168 0.188 0.135 0.151 0.1280.167 Fc OD at 10 nM   1.81 ± 1.73 ± 1.74 ± 1.75 ± 1.84 ± 2.04 ±   0.000.03 0.00 0.08 0.01 0.08 PD-1- EC50 [nM] >100 5.34 0.523 0.350 1.31 1.00his OD at 10 nM   0.30 ± 0.38 ± 1.15 ± 1.25 ± 0.69 ± 0.27 ±   0.03 0.010.04 0.02 0.00 0.02

Example 3: Antibodies isolated from phage display library blockhPD-1/hPD-L1 interactions To examine the ability of anti-hPD1 antibodies19B6, 19B10, 23A8, 23H9, 24H9, 23A11 (isolated from HEK293 cells) toblock hPD-1/hPD-L1 interactions, high-binding (Immulon 2HB) plates werecoated with human PD-L1-Fc (R&D, cat #156-B7-100) or PD-L2-Fc (R&D, cat#1224_PL), respectively, and blocked with 3% PBS milk. Serial dilutionsof antibodies 19B6, 19B10, 23A8, 23H9, 24H9, and 23A11 were mixed with0.5 μg/ml (final concentration) of biotin labeled hPD-1-Fc and incubatedat room temperature for 1 h. The mixture was transferred to the PD-L1-Fcor PD-L2-Fc coated plate and incubated at RT for additional 1-2 h. Afterwashing plate with PBST (0.05%) three times, 1/5000-1/10,000 dilutedStrepavidin-HRP (Thermofisher, cat #21140) was added to the plates,which were incubated at room temperature for about 1 h. Color wasdeveloped by adding 1:1 dilution of substrate A and B from TMB, followedby neutralizing with 1N H₂SO₄. OD₄₅₀ was read using a TECAN plate reader(Table 2). The means±S.D. of OD reading was plotted against the logantibody concentration using GraphPad Prism8. The EC50 value wascalculated by analyzing log (agonist) vs. response (three parameters) orlog(agonist) vs.—response variable slope (four parameters). The datapoints are the means±S.D. of duplicate determinations (FIG. 3 ). All sixantibodies were found to block hPD-1-Fc binding to human PD-L1 withdifferent IC50 values (Table 2 and FIG. 3 ). All six antibodies havevery similar affinity to hPD-Fc. However, antibodies 23A8, 23H9 and 24H9showed superior blocking activity to hPD-1-his compared to the otherantibodies (Table 2 and FIG. 3 ). The three lead clones 23A8, 23H9, and24H9 showed >93% inhibition and >70% inhibition in the presence of 30and 3.3 nM antibody, respectively (Table 2 and FIG. 3 ).

TABLE 2 Blocking of hPD-1 to hPD-L1 by antibodies 19B6, 19B10, 23A8,23H9, 24H9, and 23A11. Blocking ELISA 19B6 19B10 23A8 23H9 24H9 23A11IC50 [nM] 2.69 28.1 1.85 2.04 2.28 2.61 Inhibition % 85.9 ± 65.2 ± 93.1± 94.7 ± 92.9 ± 86.6 ± (30 nM) 0.8 0.6 0.2 0.14 0.1 3.0 Inhibition %51.9 ± 22.9 ± 80.5 ± 82.5 ± 70.0 ± 54.9 ± (3.3 nM) 2.1 2.4 1.0 0.4  1.17.1

Example 4: Antibodies Isolated from Phage Display Library Bind to CellsExpressing Human PD-1

Binding of anti-PD-1 antibodies to HEK293 and Jurkat cellsover-expressing human PD-1: Cells were cultured in IMDM medium withglutamine, supplemented with 10% heat-inactive fetal bovine serum (FBS).Cells were first incubated with the indicated anti-PD-1 antibodies,followed by incubation with an PE-anti human IgG1-Fc(R-phycoerythrin-conjugated goat anti-human IgG: Jackson ImmunoResearchcat #109-116-098). Antibody binding to hPD-1 was determined using a flowcytometry Guava EasyCyte™ HT Sampling Flow Cytometer (EMD Millipore).The data points in FIG. 4 were represent the mean±S.D. of duplicatedeterminations. Antibodies 23A8, 23H9, and 19B10 showed strongestbinding to HEK 293 cells and antibodies 23A8, 24H9, and 19B10 showedhighest mean fluorescence intensity (MFI) (Table 4 and FIG. 4 ).

Binding of anti-PD-1 antibodies to SEB-activated human PBMCs: PBMCs wereisolated from LeukoPak (an enriched leukapheresis containing highlyconcentrated blood cells including monocytes, lymphocytes, platelets,plasma, as well as red cells) using Histopaque-1077 (Sigma) using themanufacturer's instructions. PBMCs were cultured at 2×10⁶/mL in a12-well plate containing IMDM (Gibco, Life Technologies) supplementedwith 10% FBS (GE Healthcare HyClone) and activated by 1 ug/mL SEB(Staphylococcal enterotoxin B, Sigma) for 3 to 4 days. Activated PBMCswere first incubated with indicated anti-PD-1 antibodies, followed byincubation with a combination of PE-anti human IgG1-Fc(R-phycoerythrin-conjugated goat anti-human IgG: Jackson ImmunoResearchcat #109-116-098) and either anti human CD4-APC (eBioscience cloneRPA-T4 cat #17-0049-42) or anti human CD8-APC (eBioscience clone RPA-T8cat #17-0088-42). Antibody binding to PD-1 expressed on cells wasevaluated using a Guava EasyCyte™ HT Sampling Flow Cytometer (EMDMillipore). Data points were generated by calculating the means±S.D. ofduplicate determinations (Table 3 and FIG. 4 ). Antibodies 19B6, 23A8,23H9, and 24H9 exhibited the highest MFI and lowest EC50 for binding toPBMCs among the antibodies tested and were selected for furthercharacterization (Table 3 and FIG. 4 ).

TABLE 3 Binding of indicated anti-PD-1 antibodies to (1) hPD-1-293cells, (2) SEB-activated PBMCs from donor “EA” (low PD-1 expression),and (3) SEB-activated PBMCs from donor “AF” (high PD-1 expression).Binding to cell- expressed PD1 19B6 19B10 23A8 23H9 24H9 23A11 hPD1-293EC50 Very 16.0 0.299 2.53 58.7 Very [nM] low low MFI at 20.84 ± 45.07 ±104.85 ±  38.84 ± 59.04 ± 21.77 ± 68 nM 0.48 0.0 1.68 4.03 6.30 0.16 SEBEC50 13.6 117 1.51 0.993 1.37 19.0 activated [nM] PBMC, MFI at 13.74 ±12.41 19.88 ± 26.19 ± 18.71 ± 14.58 ± donor EA 100 nM 0.05 0.28 0.180.24 0.01 SEB EC50 10.7 104 1.89 0.829 0.913 13.4 activated [nM] PBMC,MFI at 51.23 ± 25.19 ± 80.29 ± 108.59 ± 70.52 ± 46.19 ± donor AF 100 nM1.73 0.15 1.33 5.01 3.74 1.18

Example 5: Antibodies Isolated from Phage Display Library DisruptIntercellular PD-1/PD-L1 Interactions

To assess the ability of selected anti-PD-1 antibodies to disrupt thebinding of PD-1 expressing cells to PD-L1 expressing cells, the PromegaPD-1/PD-L1 Blockade Bioassay was used. This assay consists of twogenetically engineered cell lines, PD-1 effector cells and PD-L1aAPC/CHO-K1 cells. When co-cultured, the PD-1/PD-L1 interaction inhibitsTCR-mediated luminescence. When the PD-1/PD-L1 interaction is disrupted,TCR activation induces luminescence (via activation of the NFAT pathway)that can be detected by addition of Bio-Glo™ Reagent and quantitationwith a luminometer.

The experiment was performed according to the manufacturer'sinstructions. Briefly, PD-L1 aAPC/CHO-K1 cells were plated and incubatedfor 16-20 h prior to the addition of increasing concentrations ofanti-PD-1 antibodies and PD-1 effector cells. After 6 h of incubation at37° C., Bio-Glo™ Reagent was added and luminescence (recorded asrelative light unites, RLU) was measured by using a TECAN plate reader.Fold induction was calculated by RLU (induced-background)/RLU (noantibody control-background). Data points are represented as means±S.D.of duplicate determinations (FIG. 5 ). Antibodies 19B6, 23A8, 23H9 and24H9 blocked hPD-1/hPD-L1 interaction with different EC50s and foldinduction (Table 4 and FIG. 5 ).

TABLE 4 Blocking of hPD-1/hPD-L1 interactions by antibodies 19B6, 23A8,23H9, and 24H9. 23A8 23H9 24H9 19B6 EC 50, μg/mL 4.73 0.688 0.823 2.127Fold of 2.81 ± 0.16 4.42 ± 0.11 3.96 ± 0.12 3.04 ± 0.29 induction

Antibodies 23H9 and 24H9 were selected for light chain shuffling,wherein the heavy chains of 23H9 or 24H9, respectively, were paired witha library of light chains. Antibodies 31B1, 33C4, 33G8, 34C1 (see SEQ IDNOs: 49-65, Table 25) were obtained by light chain shuffling using theheavy chain of 23H9. Antibodies 32A11, 32D11, 32D2, 32G6, 38A10, 38A1i,38A4, 38A6, 38A8, 38B1, 38B10, 38B2, 38C11, 38C6, 38G11, 38G9, 38H3,39A3, 39B11, 39B3, 39B6, 39F11, 39G5, 39G8, 39H11, 39H2, and 39H7 (seeSEQ ID NOs: 66-138, Table 25 were obtained by light chain shufflingusing the heavy chain of 24H9.

Example 6: Anti-hPD-1 Antibody 23H9 and its Derivatives Bind to hPD-1and Block hPD-1/hPD-L1 Interactions

The ability of anti-hPD1 antibody 23H9 and its derivatives 31B1, 33C4,33G8, and 34C1 to bind to hPD1 and to block hPD-1/hPD-L1 interactionswas determined using the protocols described in Examples 2 and 3,respectively. The parental antibody 23H9 and its derivatives showed nosignificant difference in binding to hPD-1-Fc and biotin-hPD-1-hiscaptured by strepavidin (Table 5 and FIG. 6A). However, all derivativeswere more potent than their parental 23H9 in binding to the anti-hiscaptured hPD-1-his and in blocking interactions of hPD-1-F/hPD-L1-Fc(Table 5 and FIG. 6B). Two clones (31B1 and 33C4) were selected forfurther characterization (Table 5).

TABLE 5 Binding of antibodies 23H9, 31B1, 33C4, 33G8, and 34C1 tohPD-1-Fc, binding to strepavidin-captured biotin-hPD-1-his, binding toahis-captured hPD-1-his, and blocking of hPD-1-Fc/hPD-L1-Fc interaction.Binding/blocking ELISA 23H9 31B1 33C4 −34C1 33G8 Binding hPD1-Fc EC50,nM 0.121 0.167 0.177 0.147 0.113 OD450 at 10 0.916 ± 0.974 ± 0.925 ±0.926 ± 0.900 ± nM 0.045 0.082 0.009 0.045 0.006 Biotin-hPD-1-his EC50,nM 0.18 0.244 0.209 0.233 0.197 captured by Strep OD450 at 10 0.890 ±0.953 ± 0.950 ± 0.945 ± 0.909 ± nM 0.004 0.006 0.069 0.014 0.028hPD-1-his EC50, nM 0.238 0.095 0.061 0.0866 0.149 captured by anti-OD450 at 10 0.406 ± 0.590 ± 0.578 ± 0.521 ± 0.487 ± his nM 0.024 0.0260.015 0.009 0.040 Blocking PD1/PD-L1 IC50, nM 1.27 2.15 2.06 2.01 1.81Inhibition % 90.4 ± 91.4 ± 92.5 ± 89.3 ± 90.5 ± at 50 nM 0.3 0.2 0.0 0.10.2 Inhibition % 88.6 ± 90.2 ± 91.2 ± 89.1 ± 89.9 ± at 5.6 nM 0.5 0.30.2 0.1 1.2

Example 7: Anti-hPD-1 Antibody 24H9 and its Derivatives Bind to hPD-1and Block hPD-1/hPD-L1 Interactions

The ability of anti-hPD1 antibody 24H9 and its derivatives 32A11, 32D2,32D11, 38A6, 38A10, 38A11, 38B2, 38C11, 39B3, 39B11, 39G8 and 38H11 tobind to hPD1 and to block hPD-1/hPD-L1 interactions was determined usingthe protocols described in Examples 2 and 3, respectively. The parentalantibody 24H9 and its derivatives showed no significant difference inbinding to hPD-1-Fc and hPD-A-his captured by strepavidin (FIG. 7 ).However, all derivatives were found to be more potent than parental 24H9in binding to the anti-his captured hPD-1-his and blocking interactionof hPD-1-Fc-hPD-L1-Fc (FIG. 7 ).

Two clones (38A6 and 38D2) were picked for further characterization(Table 6).

TABLE 6 Binding of antibodies 24H9, 32A11, 32D2, 32D11, 38A6, 38A10,38A11, 38B2, 38C11, 39B3, 39B11, 39G8 and 38H11 to hPD-1-Fc, binding tostrepavidin-captured hPD-1-his, binding to ahis captured hPD-1-his, andblocking of hPD-1/hPD-L1 interaction. Strep captured Anti-his capturedhPD-1-Fc PD-1-his PD-1-his Blocking OD at OD at OD at Inhibition NameEC50 10 nM EC50 10 nM EC50 10 nM IC50 at 50 nM 24H9 0.117 0.982 ± 0.2060.915 ± 0.738 0.207 ± 3.073 87.4 ± 0.5 0.007 0.019 0.006 32A11 0.1090.921 ± 0.21 0.919 ± 0.221 1.035 ± 2.639 91.1 ± 0.0 0.003 0.005 0.00332D2 0.084 0.922 ± 0.203 0.932 ± 0.216 0.591 ± 2.581 91.7 ± 0.1 0.0270.027 0.010 32D11 0.114 0.952 ± 0.177 0.935 ± 0.229 0.602 ± 2.418 91.9 ±0.1 0.039 0.004 0.013 38A6 0.102 0.993 ± 0.175 0.948 ± 0.217 0.576 ±2.183 91.7 ± 0.2 0.004 0.013 0.001 24H9 0.11 1.050 ± 0.272 0.994 ± 0.0430.267 ± 2.535 86.8 ± 0.4 0.018 0.035 0.028 38A10 0.112 1.057 ± 0.191.003 ± 0.117 0.552 ± 2.402 92.8 ± 1.1 0.025 0.088 0.002 38A11 0.0891.033 ± 0.204 1.069 ± 0.157 0.447 ± 2.088 93.2 ± 0.1 0.014 0.002 0.05638B2 0.079 1.051 ± 0.145 1.044 ± 0.076 0.662 ± 1.63 93.4 ± 0.0 0.0040.011 0.053 38C11 0.104 1.020 ± 0.293 1.049 ± 0.229 0.404 ± 2.384 92.7 ±0.0 0.024 0.019 0.024 24H9 0.171 1.092 ± 0.336 0.971 ± 0.797 0.165 ±2.698 86.5 ± 0.3 0.010 0.013 0.034 39B3 0.179 1.079 ± 0.264 0.963 ±5.013 0.617 ± 2.315 92.9 ± 0.1 0.009 0.008 0.061 39B11 0.116 1.082 ±0.234 0.990 ± 23.98 0.482 ± 2.023 93.3 ± 0.0 0.011 0.001 0.014 39G80.099 1.063 ± 0.224 0.981 ± 4.982 0.484 ± 1.732 93.2 ± 0.0 0.019 0.0190.006 38H11 0.115 1.083 ± 0.235 0.956 ± 2.146 0.699 ± 2.05 93.6 ± 0.10.018 0.014 0.022

Example 8: The Binding Profile of 23H9 and 24H9 Derivatives Indicatethat these Antibodies Bind to Different Binding Epitopes

Dose response binding assay using 23H9, 31B1, 33C4, 24H9, 38A6, and 38B2were performed according to the protocol disclosed in Example 4. Theresults are shown in FIG. 8 . 23H9 derivatives and 24H9 derivatives werefound to be more potent in binding to PD-1 expressing cells than theirparental antibodies (Table 7). Further, 23H9 derivatives and 24H9derivatives showed different binding profiles, indicating indicated theybind to different binding epitopes (FIG. 8 ).

TABLE 7 Binding of indicated antibodies to hPD-1-293 and hPD-1 Jurkat,and SEB-activated PBMC from donor “FA” and “FB”. 23H9 31B1 33C4 24H938A6 38B2 hPD1-293 EC50, nM 0.33 0.077 0.075 2.69 0.15 0.12 MFI at 543.01 46.4 ± 48.2 ± 42.79 110.3 ± 118.3 ± nM 0.2 1.6 2.5 0.3 hPD1- EC50,nM 0.42 0.084 0.084 0.77 0.21 0.16 Jurkat MFI at 5 119.6 91.6 ± 98.3 ±80.6  89.5 ±  91.0 ± nM 6.7 7.5 3.9 0.0 Activated EC50, nM NA 0.0490.041 NA 0.21 0.15 PBMC- MFI at 25 NA 18.4 ± 17.6 ± NA  14.5 ±  16.6 ±(FA) nM 0.6 0.2 0.0 0.2 Activated EC50, nM NA 0.025 0.050 NA 0.17 0.10PBMC- MFI at 25 NA 17.8 ± 18.7 ± NA  15.9 ±  16.7 ± (FB) nM 0.3 0.4 0.51.7

Example 9: Antibodies 23H9, 24H9 and their Derivatives DisruptIntercellular PD-1/PD-L1 Interactions

A PD-1 blockade bioassay was performed using antibodies 23H9, 31B1,33C4, 24H9, 38A6, and 38B2 according to the protocol outlined in Example5. All derivatives were found to be more potent than their parentalantibody 23H9 and 24H9 in blocking hPD-1-hPD-L1 interactions (FIG. 9 ).38B2 and 31B1 were found to show lowest EC50 and maximum fold inductionamong the six antibodies tested (Table 8 and FIG. 9 ).

TABLE 8 Blocking of PD-1/PD-L1 interactions by antibodies 23H9, 31B1,33C4 and 24H9, 38B2, and 38A6 using a Promega binding assay. 23H9 31B133C4 24H9 38A6 38B2 EC50, μg/ml 0.551 0.139 0.165 0.855 0.164 0.154 Foldinduction 5.58 ± 6.48 ± 6.24 ± 4.97 ± 5.66 ± 6.54 ± 0.28  0.035 0.0920.049 0.636 0.071

Example 10: The Effect of Anti-PD-1 Antibodies on Cytokine Production inSEB-Activated PBMCs

The effect of anti-PD-1 antibodies on cytokine production using 38A6,38B2, 31B1, and 33C4 in SEB activated PBMC from donor FA was assessed bymeasuring the amount of cytokines released by PBMCs in the culturemedium. PBMCs were isolated from LeukoPak (an enriched leukapheresiscontaining highly concentrated blood cells including monocytes,lymphocytes, platelets, plasma, as well as red cells) usingHistopaque-1077 (Sigma) per manufacture instruction. PBMCs were culturedat 1×10⁵ per well in 96 well plate containing IMDM (Gibco, LifeTechnologies) supplemented with 10% FBS (GE Healthcare HyClone). Thecells were activated by 0.01 ug/mL SEB (Staphylococcal enterotoxin B,Sigma) for 2 days. Supernatants were collected for the measurement ofIL-2 using Duoset ELISA Kit (R&D Systems) per the manufacturer'sinstructions (FIG. 10A).

The experiment was repeated to obtain EC50 values for antibodies 23H9,31B1, 24H9, and 38B2 using PBMCs from donor EA (FIG. 10B).

IL-2 concentration increased in the presence of all antibodies in adose-dependent manner (FIGS. 10A, 10B). IL-2 concentrations increased inpresence of 23H9 derivative 31B1 and 24H9 derivative 38B2, which werefound to be more potent than their parental antibody 23H9 and 24H9,respectively (Table 9 and FIG. 10B).

TABLE 9 IL-2 accumulation for PBMCs from donor EAand respective EC50 andIL-2 accumulation data in presence of antibodies 23H9, 31B1, 24H9, and38B2. 23H9 31B1 24H9 38B2 EC 50, nM 0.344 0.107 >10 0.019 IL-2, pg/ml in3931 ± 52 5809 ± 90 4476 ± 35 4995 ± 140 10 nM

Example 11: Biacore Analysis of Selected 23H9 and 24H9 Derivatives

The binding kinetics of the antibodies to either hPD-1-Fc or hPD-1-hiswas measured by surface plasmon resonance (SPR) using the Biacore T200.Human PD-1-Fc or PD-1-his protein was immobilized at pH5 onto a Series SCM5 sensor chip using standard amine coupling chemistry. Antibodies 23H9and 23H9 derivatives 31B1, 33C4 as well as 24H9 and 24H9 derivatives38A6, 38B2, 32D11, 39G8, 38A10, and 38A11 were injected at 30 μl/min atconcentrations ranging from 1.5 to 100 nM over the immobilized surfaceusing 1×HBSEP as the running buffer. The contact time (associationphase) was 3 mins. The dissociation time was 6-10 mins. Regeneration wasperformed after each binding cycle with an injection of 20 mM HCl for 30sat 30 μl/min flow rate. Sensorgrams were obtained at each concentrationand the derived curves were fit to a 1:1 Langmuir binding model usingBiaevaluation software. Derivative 31B1 and 33C4 were found to be morepotent than the parental 23H9 in their binding to hPD-1-Fc (Table 10).All derivatives of 24H9 were found to be more potent than the parentalantibody 24H9 in their binding to hPD-1-his and hPD-1-Fc.

TABLE 10 hPD1 binding characteristics of indicated antibodies asdetermined by SPR analysis. hPD1his hPD1Fc Antibody k_(a) k_(d) K_(D)k_(a) k_(d) K_(D) 24H9 1.64 E+05 2.27 E−03 1.39 E−08 3.97 E+05 5.81 E−041.46 E−09 38B2 1.26 E+05 4.01 E−05 3.1S E−10 9.40 E+05 2.34 E−04 2.49E−10 38A6 4.72 E+05 5.59 E−04 1.18 E−09 2.01 E+05 9.57 E−05 4.77 E−1032D11 1.67 E+05 2.41 E−04 1.44 E−09 1.54 E+05 8.52 E−05 5.54 E−10 39G81.81 E+05 4.67 E−04 2.58 E−09 2.15 E+05 1.71 E−04 7.94 E−10 38A10 1.12E+05 5.41 E−04 4.82 E−09 2.06 E+05 1.72 E−04 8.34 E−10 38A11 1.07 E+055.25 E−04 4.93 E−09 2.20 E+05 2.25 E−04 1.02 E−09 hPD1his hPD1Fc hPD1hishPD1Fc hPD1his hPD1Fc Antibody k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) 23H98.64 E+04 4.24 E−04 4.91 E−09 1.69 E+05 1.57 E−04 9.28 E−10 31B1 2.78E+05 1.82 E−03 6.56 E−09 3.44 E+05 2.37 E−04 6.88 E−10 33C4 9.43 E+046.35 E−04 6.73 E−09 2.77 E+05 1.60 E−04 5.75 E−10

Example 12: Thermo-Stability Measurement of Selected 23H9 and 24H9Derivatives by Differential Scanning Calorimeter (DSC)

Selected 23H9 and 24H9 derivatives, 31B1, 33C4, 38A6, and 38B2 werecloned and expressed by WuXi Biologics by using WuXi's mammalianexpression vector and CHO knockout cell line. All antibodies werepurified using protein A and SEC-HPLC. More than 2.5 mg of purifiedantibody was obtained from 20 ml culture after Protein A and SEC-HPLCpurification. The monomer percentage of all antibodies was over 96% withendotoxin levels lower than 1.0 EU/mg. Differential scanning calorimetry(DSC) was used to determine the stability of the four antibodies. TheDSC analysis was performed using a Malven DSC system. The protein samplewas first diluted to 1 mg/mL with formulation buffer before analysis.400 μl respective formulation buffer was added to a 96-well plate asreference and 400 μL protein sample was added. The samples were heatedfrom 10° C. to 100° C. at a heating rate of 90° C./h in the capillaryDSC system. The DSC results (T_(m) onset and T_(m) values) were analyzedusing the vendor's software. The lowest thermal transition midpoint(T_(m1)) of 31B1, 33C4, 38A6, and 38B2 were 70.5, 74.0, 71.0, and 71.9°C. respectively (FIG. 11A). The monomer percentage of all antibodies wasover 96% with Endotoxin level lower than 1.0 EU/mg (Table 11). 33C4 and38B2 were found to be most thermostable of all the antibodies tested(Table 11).

TABLE 11 Thermo-stability measurement for antibodies 31B1, 33C4, 38A6,and 38B2 as determined by differential scanning calorimeter (DSC) andantibody monomer percentage after SEC-HPLC purification. Protein A SECHPLC Endotoxin Amount Purity, Amount Purity, DSC (° C.) (EU/mg, Name(mg) % (mg) % T_(m on) T_(m1) T_(m2) T_(m3) LAL) 780 38 4.70 97.2 2.4496.1 60.8 70.5 74.0 83.7 <1.0 (31B1) 780 39 4.10 95.8 2.43 96.9 62.174.0 83.8 ND <1.0 (33C4) 780 46 7.95 94.6 5.22 98.7 62.8 71.0 77.6 84.9<1.0 (38A6) 780 54 7.24 95.6 5.24 99.3 62.6 71.9 84.0 ND <1.0 (38B2)

Example 13: Selected 23H9 and 24H9 Derivatives Bind to hPD-1 and BlockhPD-1/hPD-L1 as Well as hPD-1/hPD-L2 Interactions

Binding and Blocking ELISA were performed using antibodies 31B1, 33C4,38A6, 38B2 isolated from transient CHOk cells using the protocolsoutlined in Examples 2 and 3, respectively. Comparison of the antibodiesusing binding ELISA showed no significant differences among thederivatives in binding to hPD-1-Fc, strepavidin-captured biotin-labeledhPD-1his and anti-his antibody captured hPD-1-his directly bound to theplates (FIG. 12A-C). However, EC50s of 38B2 and 38A6 (both 24H9derivatives) were lower than 31B2 and 33C4 (both 23H9 derivatives) andwere superior to both controls (Table 12 and FIG. 12A-C).

Comparison of the antibodies using blocking ELISAs showed no significantdifferences among the derivatives in blocking hPD-1/hPD-1 andhPD-1/hPD-L2 interactions (FIG. 12D-E). However, similar to bindingELISA, antibodies 38A6 and 38B2 (both 24H9 derivatives) were found to bemore potent than 31B2 and 33C4 (both 23H9 derivatives), especially forthe inhibition of hPD-L2 binding to hPD-1 (Table 13 and FIG. 12D-E).

TABLE 12 Binding of antibodies 31B1, 33C4, 38B2, and 38A6 to hPD-1FC,streptavidin-captured biotin-labeled hPD-lhis, and anti-his antibodycaptured hPD-1his. Ec50 nM ctB ctM 31B1 33C4 38A6 38B2 PD-1-Fc 0.1020.166 0.114 0.128 0.079 0.096 strep/Bio-PD-1-his 0.084 0.15 0.104 0.1310.084 0.081 anti-his/PD-1h-is 0.094 0.105 0.139 0.142 0.089 0.060 ctB =control B (Nivolumab, sold under the brand name Opdivo). ctM = control M(Pembrolizumab, sold under the brand name Keytruda).

TABLE 13 Ability of indicated antibodies to block hPD-1-hPD-L1 andhPD-1-hPD-L2 interactions. Blocking ctB ctM 31b1 33C4 38A6 38B2 PD-IC50, nM 1.811 2.024 2.65 2.77 ~2.14   2.37 L1/ Inhibition,   50 nM 93.4± 93.1 ± 9.28 ± 93.3 ± 92.2 ± 93.9 ± PD-1 % 0.3 0.5 0.3 0.1 0.0 0.1 5.56nM 92.3 ± 90.3 ± 89.7 ± 88.9 ± 92.0 ± 92.7 ± 0.4  0.22 0.1 0.4 0.8 0.6PD- IC50, nM 2.222 2.749 3.94 3.3  2.39 1.93 L2/ Inhibition,  100 nM93.8 ± 94.8 ± 94.5 ± 94.6 ± 94.8 ± 95.1 ± PD-1 % 0.1 0.1 0.1 0.3 0.1 0.0 3.7 nM 88.6 ± 85.8 ± 41.4 ± 53.4 ± 90.4 ± 93.9 ± 0.3 4.0 0.3 5.7 0.80.3 ctB = control B (Nivolumab, sold under the brand name Opdivo). ctM =control M (Pembrolizumab, sold under the brand name Keytruda).

Example 14: Dose-Response Binding Assay Using 33B1, 33C4, 38A6, and 38B2

Dose response binding assays were performed using antibodies 33B1, 33C4(both 23H9 derivatives), 38A6 and 38B2 (both 24H9 derivatives) using theprotocols outlined in Example 4.

Dose-response binding assays showed no difference between the twoderivatives from the same parental antibody in binding of the antibodiesto hPD-1 transfected HEK-293 and Jurkat cells, and SEB-activated PBMCs(Table 14 and FIG. 13 ). However, the derivatives derived from differentparental antibodies showed differences of binding to different celllines, suggesting that 23H9 derivatives and 24H9 derivatives havedifferent binding epitopes (Table 14 and FIG. 13 ).

TABLE 14 Dose-response binding of antibodies 31B1, 33C4, 38A6, and 38B2to hPD-1-293 cells, hPD-1-Jurkat cells, SEB-activated PBMCs from donorFA, and SEB-activated PBMCs from donor FB. ctB ctM 31B1 MFI, MFI, 5 MFI,Cell line EC50 5 nM EC50 nM EC50 5 nM hPD-1-Jurkat 0.101 64.4 ± 0.08558.8 ± 3.1 0.100 67.0 ± cells 1.2 2.1 hPD-1-293 cells 0.156 195.1 ± 0.141 223.4 ± 10.0 0.229  186 ± 15.7  2   SEB activated 0.084 61.6 ±0.082  78.6 ± 5.73 0.136 98.5 ± PBMC, donor 8.1 10.1  FA SEB activated0.151 55.5 ± 0.102 50.6 ± 0.8 0.209 67.7 ± PBMC, donor 4.9 0.2 FB 33C438A6 38B2 MFI, MFI, MFI, Cell line EC50 5 nM EC50 5 nM EC50 5 nMhPD-1-Jurkat 0.115 65.2 ± 0.451 59.9 ± 1.3 0.362 60.8 ± cells 0.8 3.4hPD-1-293 cells 0.213  179 ± 0.211 227 ± 6  0.239  297 ± 7   2  SEB-activated 0.080 77.8 ± 0.206 65.2 ± 1.0 0.224 64.2 ± PBMCs, donor2.4 2.7 FA SEB-activated 0.174 66.9 ± 0.387 54.7 ± 2.9 0.287 55.3 ±PBMCs, donor 0.1 5.4 FB ctB = control B (Nivolumab, sold under the brandname Opdivo). ctM = control M (Pembrolizumab, sold under the brand nameKeytruda).

Example 15: Selected 23H9 and 24H9 Derivatives Bind to hPD-1 with HighAffinity

Binding of selected antibodies to hPD-1 his and hPD-1Fc was determinedby SPR. Biacore analysis of 31B1, 33C4, 38A6, and 38B2 was performedusing the protocol described in Example 11. All four antibodies werefound to have high affinity to both hPD-1-Fc and hPD-1-his (Table 15 andFIG. 14 ). However, between two derivatives of 23H9, 3 3B1 was found tobe more potent than 33C4 in both binding to PD-1-Fc and PD-1-his (Table15). Between two derivatives of 24H9, 38B2 was found to be slightly morepotent than 38A6 in binding to hPD-1-Fc and showed no significantdifference in binding to hPD-1-his. Both 31B1 and 38B2 were found to besuperior to the two controls (Table 15).

TABLE 15 Binding of antibodies 38B2, 38A6, 33C4, and 31B1 to hPD-1 hisand hPD-1Fc as determined by SPR. KD, nM KD, nM KD, nM KD, nM Kp range,(Exp. 1) (Exp. 2) (Exp. 3) (Exp. 4) nM hpD1- hPD1- hpD1- hPD1- hpD1-hPD1- hpD1- hPD1- hPD1- hPD1- Name his Fc his Fc his Fc his Fc his FcctB 2.16 0.188 <0.857 <0.166 <1.39 <0.255 0.914 NA 0.86- 0.17- 2.16 0.26ctM 3.51 0.225 <1.39 <0.345 <1.39 <0.322 1.32 NA 1.32- 0.23- 3.51 0.3531B1 <0.036 <0.014 <0.01 NA 0.353 <0.01 <0.019 NA 0.01- 0.01- 0.35 0.0233C4 0.704 <0.022 0.205 <0.01 0.274 <0.01 <0.054 NA 0.05- 0.01- 0.700.02 38A6 2.3 <0.188 <0.586 <0.136 <0.574 <0.301 <0.090 NA 0.09- 0.14-2.30 0.30 38B2 0.946 <0.033 0.423 <0.01 0.932 <0.01 0.205 NA 0.21- 0.01-0.95 0.03 ctB = control B (Nivolumab, sold under the brand name Opdivo).ctM = control M (Pembrolizumab, sold under the brand name Keytruda).

Example 16: Antibodies 31B1 and 38B2 Disrupt Intercellular PD-1/PD-L1Interactions 31B1 and 38B2

To assess the ability of 31B1 and 38B2 to disrupt the binding of PD-1expressing cells to PD-L1 expressing cells, Promega PD-1/PD-L1 BlockadeBioassays were performed using the protocol described in Example 5. Nosignificant difference was observed in EC50 and fold induction between31B1 and 38B2 (FIG. 15 ). Both 31B1 and 38B2 were found to be superiorto control B (Nivolumab, sold under the brand name Opdivo) and similarto control M (Pembrolizumab, sold under the brand name Keytruda) (Table16).

TABLE 16 Ability of antibodies 31B1 and 38B2 to blockintercellularhPD-1-hPD-L1 interactions. 31B1 38B2 ctM ctB Reading, 10 mins Exp. 1Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 EC50, μg/ml 0.168 0.172 0.189 0.1350.109 0.187 Fold induction 7.44 ± 8.40 ± 7.61 ± 8.31 ± 7.40 ± 6.86 ± at12 μg/ml 0.18 0.46 0.15 0.58 0.31 0.16 ctB = control B (Nivolumab, soldunder the brand name Opdivo). ctM = control M (Pembrolizumab, sold underthe brand name Keytruda).

Example 17: The Effect of 31B1 and 38B2 on Cytokine Production in SEBActivated PBMC

The effect of 31B1 and 38B2 on cytokine production in SEB-activatedPBMCs was determined using the protocol outlined in Example 4. 38B2 wasfound to be more potent than 31B1 and the two control antibodies tested(Table 17 and FIG. 16 ).

TABLE 17 Effect of PD-1 antibodies 31B1 and 38B2 on IL-2 cytokineproduction in SEB-activated PBMC from donor EA and FA. ctM ctB 31B1 38B2Donor Donor Donor Donor Donor Donor Name EA EA EA FA EA FA EC50, nM0.210 0.548 0.297 1.362 0.101 0.070 IL-2 (ng/mL) at 7.21 ± 6.79 ± 7.79 ±11.72 ± 7.77 ± 14.14 ± 10 nM 0.35 0.64 0.26 2.8  0.13 0.37 IL-2 (ng/ml)at 7.25 ± 6.31 ± 7.73 ± 12.16 ± 8.05 ± 13.80 ± 2.5 nM 0.08 0.05 0.030.00 0.29 0.75 ctB = control B (Nivolumab, sold under the brand nameOpdivo). ctM = control M (Pembrolizumab, sold under the brand nameKeytruda).

Example 18: Isolation of Antibodies that Cross-React with Human andMouse from Distributed Bio SuperHuman 2.0 Inc. Library

PD-1 antibodies that bind to both murine and human PD-1 were isolatedusing mPD-1-Fc (mouse IgV domain to fuse to the N-terminus of humanIgG1Fc; present as dimer in solution) or mPD-1-his (mouse IgV domain ofPD-1 connected to N-terminus of six histidine; present as monomer insolution) using the protocol outlined in Example 1.

Derivatives of anti-mPD-1 antibody R3A9 (SEQ ID NOs:139-146) by lightpair shuffling using the VH chain of R3A9. R3A9 and its derivatives(see, e.g., SEQ ID NOs: 147-167) were select to develop further. Theantibodies were cloned and expressed by WuXi Biologics using Wuxi'smammalian expression vector and CHO knockout cell line. The antibodieswere purified by protein A and SEC-HPLC. More than 2.5 mg purifiedantibodies are obtained from 20 ml culture after Protein A purification.Antibodies further purified by SEC-HPLC.

The biological properties of selected antibodies generated in accordancewith the methods of this Example are described in detail in the Examplesset forth below. FIGS. 1 and Table 25 provide the amino acid sequencesof the heavy and light chain variable regions and CDRs of selectedanti-mPD-1 antibodies (Kabat numbering).

Example 19: Anti-mPD-1 Antibody R3A9 and its Derivatives Bind to mPD-1and Block mPD-1/mPD-L1 Interactions

Dose Response Binding and Blocking ELISA were performed using R3A9 andits derivatives using the protocols as outlined in Examples 2 and 3.Binding of antibodies to mPD-1 expressing cells was performed asdescribed in Example 4. Blockage of intercellular PD-1/PD-L1interactions was examined using the Promega Bioassay described inExample 5. The results are illustrated in FIG. 17 .

The R3A9 derivative m3A7 was found to be more potent than the parentalR3A9 antibody in both binding to the soluble cell expressed mPD-1 andblocking mPD-1/mPD-L1 interactions (Table 18).

TABLE 18 Dose response binding of antibodies m3A7 and R3A9 to mPD-1- Fc,streptavidin-captured mPD-1FC, streptavidin-captured mPD- 1his, andcells expressing mPD-1; blocking of mPD-1 to PD-L1 by ELISA and blockageof intercellular ineractions between mPD-1 to PD-L1 as determined by aPromega assay. RMP1-14 is a control, monoclonal antibody reacts withmouse PD-1. M3A7 R3A9 EC50/IC50 Description (7115C) (7115A) RMP1-14 EC50, nM mPD-1-Fc 0.834 14.6 EC 50, nM Strep captured 0.187 0.156 mPD-1-FcEC 50, nM Strep captured 0.251 1.61 mPD-1-his EC 50, nM mPD-1-293 0.2280.566 IC 50, nM Blocking of mPD-1/PD-L1 0.102 0.441 0.635 interaction(ELISA) IC 50, μg/ml Blocking of intercellular 0.0709 1.625 5.594mPD-1/PD-L1 interaction

Example 20: R3A9 Derivatives m2C1, m2A1, m4D1 and m3A7 Bind to BothHuman and Murine PD-1

Dose response Binding and Blocking ELISAs for antibodies were performedusing the protocols outlined in Examples 2 and 3, respectively. Theexperiments showed all R3A9 derivatives bound strongly to both hPD-1 andmPD-1 (FIGS. 18A-C) and blocked hPD-L1-hPD-1 and mPD-L1/mPD-1interaction (FIG. 18D-E). Table 19 provides EC50/IC50 data for theantibodies tested.

TABLE 19 Binding of antibodies 38B2, m2C1, m2A1, m4D1, m3A7, and R3A9 tohPD-1, mPD-1, and streptavidin-captured biotin mPD-1, and blocking ofmPD-1/mPD-L1 or hPD1/hPDL1 interactions by antibodies 38B2, m2C1, m2A1,m4D1, m3A7, and R3A9. EC50/ hPD1 mPD1 IC50, Ab parental nM Target (38B2)m2C1 m2A1 m4D1 m3A7 (R3A9) EC50, hPD1 0.125 0.1494 0.1465 0.2208  0.63023.246 nM EC50, mPD1 No 0.5715 0.3039 2.032   0.3495 1.356 nM bindingEC50, strep No 0.2912 0.1404 1.15   0.2681 0.462 nM capt. bindingBiotin- mPD1 IC50, mPD1/ No 0.4641 0.442  0.8818  0.5373 0.722 nM mPDL1binding IC50, hPD1/ 4.25  5.707  6.01  6.02  37.77  No nM hPDL1 binding

Example 21: R3A9 Bind to Cells Expressing Human or Mouse PD-1

Binding of R3A9 and its R3A9 derivatives m2A1, m2C1, m4D1, m3A7 to PBMCscells were tested using the protocol outlined in Example 4. Further,Biacore analysis was performed on all the antibodies disclosed in thisExample using the protocol provided in Example 11.

Only three derivatives, m4d1, m2AM, and m3CM, were found to bind to bothhuman and mouse PD-1 expressed on cells (Table 21 and FIG. 19 ). Basedon cell binding (Table 20) and Biacore analysis (Table 20), m4D1 wasfound to show improved binding properties as compared to the other twoantibodies tested (Table 20 and FIG. 19 ).

TABLE 20 Binding affinity data for indicated antibodies as determined bySPR. Biacore Analysis mPD1-his mPD1-Fc hPD1-Fc k_(a) k_(d) K_(D) k_(a)k_(d) K_(D) k_(a) k_(d) K_(D) Name (1/Ms) (1/s) (M) (1/Ms) (1/s) (M)(1/Ms) (1/s) (M) 38B2 no binding no binding 2.21E+05 3.26E−05 1.47E−10m2A1 3.75E+05 1.07E−03 2.83E−09 3.33E+05 9.05E−04 2.73E−09 2.40E+054.01E−04 1.67E−09 m2C1 2.15E+05 6.93E−04 3.22E−09 2.00E+05 5.83E−042.91E−09 1.26E+05 3.55E−04 2.81E−09 m4D1 7.00E+04 2.01E−04 2.87E−096.63E+04 1.79E−04 2.69E−09 5.83E+04 6.36E−05 1.09E−09 m3A7 2.72E+058.23E−04 3.03E−09 2.61E+05 6.59E−04 2.53E−09 1.71E+05 1.76E−02 1.03E−07m3A7 2.41E+05 4.79E−04 1.98E−09 2.19E+05 4.06E−04 1.86E−09 1.32E+052.63E−03 1.99E−08 R3A9 4.09E+05 6.41E−03 1.57E−08 1.16E+05 1.40E−031.21E−08 no binding

TABLE 21 Binding of antibodies m2A1, m2C1, m4D1, R3A9, m3A7 and 38B2 tocells expressing hPD-1 or mPD-1. EC50 of cells expressing h/mPD1, nMName hPD1-293 hPD1-Jurkat mPD1-293 mPD1-eff 38B2 0.267 0.137 ND ND m2A10.987 ND 0.433 0.253 m2C1 1.263 very weak 0.411 0.373 m4D1 1.48 0.7381.139 0.441 m3A7 ND ND 0.609 0.27 m3A7 * NA NA NA N R3A9 ND ND 0.3140.645

Example 22: Promega (h/m)PD-1/(h/m)PD-L1 Blockade Bioassay Comparingm4D1 with 38B2 and m3A7

Promega blockade bioassays were performed to compare the potency ofm4D1, m3A7 and 38B2 to disrupt intercellular PD-1/PD-L1 interactionsusing the protocol outlined in Example 4. The results are depicted inFIG. 20 .

m4D1 was found to block both hPD-1/PD-L1 and mPD-1/mPD-L1 interactions.Further, m4D1 was found to be as potent as m3A7 in mPD-1/mPD-L1 blockadeassay and 3 folds less potent than 38B2 (FIG. 20 ).

Example 23: Fusion Proteins Comprising Anti-mPD1- or Anti-hPD-1Antibodies, IL-15 and, IL-15Rα Sushi Domain

Anti-mPD-1 fusion antibodies comprising antibody m3A7 (referred to asm3A7/IL-15) were engineered by appending the sushi-domain of IL-15Rα andan IL-15 polypeptide to either the C-terminus or N-terminus of theantibody. The fusion of the IL-15Rα sushi domain along with a portion ofthe hinge region, linker 1, and IL-15 is called “SD15.” See, for anexample of an SD15 domain, SEQ ID NO:174 in Table 25, SD15 domainhighlighted in bold.

In the first version of the fusion protein, the SD15 domain was fused tothe C-terminus of only one of the IgG1 CH1-CH2-CH3 domain (designated“1C-m3A7/IL-15”).

In a second version, the SD15 domain was fused to the C-termini of bothIgG1 CH1-CH2-CH3 domains (designated “2C-m3A7/IL-15”).

In a third version of the fusion protein, the SD15 domain was linked tothe N-terminus of only one of the VH domains via a second linker(designated “1N-m3A7/IL-15”).

In a fourth version of the fusion protein, the SD15 domain was linked tothe N-terminus of both of the VH domains via a second linker (designated“2N-m3A7/IL-15).

In the four constructs above, the light chains were that of aconventional antibody. Amino acid substitutions, L234A, L235A, P329G(LALA-PG) were introduced in both the CH2 domains for eliminatingcomplement binding and fixation as well as Fc-7 dependentantibody-dependent cell-mediated cytotoxity (ADCC).

Further, for heterodimeric fusions, mutations were introduced intoconstant regions of the heavy chains, wherein the amino acidsubstitutions were selected from one of the following groups: (1)CH3-1:T366Y (“knob”); CH3-2: Y407T (“hole”), (2) CH3-1: S354C, T366W(“knob”); CH3-2: Y349C, T366S, L368A, Y407V (“hole”) and (3) CH3-1:T350V, L351Y, F405A, Y407V (“knob”); CH3-2: T350V, T366L, K392L, T394W(“hole”).

The fusion antibodies were expressed transiently in CHOk cells. At least95% purity of the monomer was obtained after purification with Protein Aand SEC-HPLC. Four corresponding versions of anti hPD-1/IL-15 fusionantibodies comprising antibody 38B2 (described herein as 38B2-hSD15 or38B2/IL-15)—1C-38B2-hSD15, 2C-38B2-hSD15, 1N-38B2-hSD15,2N_-38B2-hSD15—were created following the same procedure. Threecorresponding versions of 1N_38B2-SD15 bearing an amino acidsubstitution in position 65 of the IL-15 sequence, 65A_1N_38B2-hSD15,65D_1N_38B2-hSd15, and 65S_1N_38B2-hSD15 were created using the sameprocedure.

Four corresponding versions of control fusion antibodies (DP47/IL-15)1C-DP47/IL-15, 2C-DP47/IL-15, 1N-DP47/IL-15, 2N-DP47/IL-15 were createdfollowing the same procedure. DP47 was used as a non-targeting controlantibody. Schematic diagram showing the antibody orientations are shownin FIG. 21 .

Example 24: mPD-1/IL-15 Fusion Molecules Bind to Murine PD-1 and BlockInteractions Between mPD-1 and mPD-L1

The ability of fusion proteins 1N-m3A7/IL-15, 1C-m3A7/IL-15,2N-m3A7/IL-15, and 2C-m3A7/IL-15 to bind to mPD-1 and to blockinteractions between mPD-1 and mPD-L1 was assessed by ELISA, followingthe protocols described in Example 2 and 3, respectively.

No significant differences in binding and blocking capacity wereobserved among the four different molecules (Table 22 and FIG. 22 ).

TABLE 22 Binding to mPD-1 and blocking of mPD-L1 to mPD-1 by fusionproteins 1N_m3A7/IL-15, 2N_m3A7/IlL5, 1C_m3A7/IlL5, and 2C_m3A7/IL-15.Name Ka Kd KD Binding to mPD-1-his 2C-m3A7/IL15 2.00E+05 4.94E−052.47E−10 2N-m3A7/IL15 9.21E+04 2.61E−05 2.83E−10 1C-m3A7/IL15 2.32E+056.87E−05 2.96E−10 1N-m3A7/IL15 1.52E+05 6.95E−05 4.57E−10 Binding tomPD-1-Fc* 2N-m3A7/IL15 1.15E+05 1.05E−06 9.09E−12 2C-m3A7/IL15 1.81E+051.08E−05 5.93E−11 1N-m3A7/IL15 1.46E+05 1.28E−05 8.78E−11 1C-m3A7/IL152.00E+05 1.86E−05 9.27E−11 *The off rate was too slow and outside themachine limit.

Example 25: Effect of mPD-1/IL-15 Fusion Molecules on IL-2-DependentCell Growth Stimulation of Mouse Lymphocytes CTLL2 and C57BL6 SpleenCells

CTLL2 Culture and Proliferation Assay: The murine T lymphocyte cell lineCTLL2 was cultured in IMDM medium supplemented with 10% heat-inactivatedFBS and 10% IL-2 supplement (T cell culture supplement with ConA,Corning). The following fusion proteins were tested in this example:1N-m3A7/IL-15, 2N-m3A7/IL-15, 1C-m3A7/IL-15 and 2C-m3A7/IL-15.1N-DP47/IL-15, 2N-DP47/IL-15, 1C-DP47/IL-15, 2C-DP47/IL-15 and m3A7 wereused as controls.

Serial dilutions of antibodies or fusion proteins were added in a96-well plate, and then cells were seeded at 2.5×10⁴ per well in IMDMmedium supplemented with 10% heat-inactivated FBS. The cells wereincubated at 37° C. for 3-5 days and cell proliferation was detectedusing CellTiter-Glo® luminescent cell viability assay kit (Promega).

Mouse Spleen Cell Isolation and Proliferation Assay: C57BL/6 mousespleens were homogenized in IMDM medium through the 70 μm cell strainer.1×RBC lysis buffer (eBioscience) was added to spleen cells and left for3 min with occasional shaking, and then diluted with 5-fold volume ofPBS buffer. After washing with PBS buffer, the splenocyte cellsuspension was prepared in complete IMDM medium supplemented with 10%heat-inactivated FBS for proliferation assay immediately. The followingfusion proteins were tested in this example: 1N-m3A7/IL-15,2N-m3A7/IL-15, 1C-m3A7/IL-15 and 2C-m3A7/IL-15. 1N-DP47/IL-15,2N-DP47/IL-15, 1C-DP47/IL-15, and 2C-DP47/IL-15 were used as controls.

Serial dilutions of the fusion proteins and controls were added in a96-well plate and then cells were seeded at 1×10⁵ per well. The cellswere cultured for 5-7 days at 37° C. and collected for flow-cytometryanalysis. The cells were washed and stained with fixable viability dyeFluor780 (eBioscience) for 30 min at 4° C. The cells were fixed afterwashing and were permeabilized with Foxp3 transcription factor stainingbuffer set (eBioscience), followed by staining with CD3(145-2C11)-PE,CD4(RM4-5)-APC, CD8α-(53-6.7)-PECy7 and Ki67(SolA15)-FITC fromeBioscience. Proliferative CD4 (CD4⁺Ki67⁺) or CD8 (CD8⁺Ki67⁺) T cellpopulation were analyzed and defined using Guava Flow Cytometry EasyCytesystem.

All mPD-1/IL-15 fusion constructs stimulated both IL-2 dependent mouselymphocyte CTLL2 (FIG. 23A) and C57BL6 spleen cell growth (FIG. 23B).Analysis of T cell populations showed that only the percentage of CD8⁺ Tcell population increased while the population percentage of CD4⁺ Tcells remained unaffected (FIG. 23C). Out of the four constructs tested,1N-m3A7/IL-15 showed the least stimulatory effect on IL-2 dependentmouse lymphocyte and spleen cell growth and T cell proliferation, while2C-m3A7/IL-15 showed the most stimulatory effect on the cell growth andT cell proliferation.

Example 26: Efficacy Study of mPD-1/IL-15 Fusion Molecules in PD-1/PD-L1Lewis Lung Cancer Model

To determine if the orientation or the number of IL-15/IL-15Rα sushidomains in a fusion protein have any effect on the ability of the fusionmolecules to reduce tumor growth, an in vivo efficacy study in an LL/2Murine Lung Cancer Syngeneic Model was conducted by Crown Bioscience(Taicang) using the m3A7/IL-15/IL-15Rα sushi fusion proteins Inc.Constructs 1N-m3A7/IL-15 and 1C-m3A7/IL-15 were tested in this Example,while 1N-DP47/IL-15 and 1C-DP47/IL-15 were used as controls.

C57BL6 strain mice, age from 8 to 10 weeks and weight from 16.5 to 20.7g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd(Shanghai, China). The LL/2 tumor cells were maintained in vitro withDMEM medium supplemented with 10% fetal bovine serum at 37° C. inpresence of 5% CO2. The cells were harvested in exponential growth phaseand counted before tumor inoculation. The fusion proteins used in thisexample were produced by WuXi Biologics and exhibited at least 95%purities and contained more than 95% monomer.

Each mouse was inoculated subcutaneously at the right rear-flank regionwith LL/2 tumor cells (3×10⁵) in 0.1 ml of PBS for tumor development.The date of randomization and the first dosing day was denoted as day 0.When the mean tumor size reached approximately 100 mm³ the randomizationprocess was started. Mice were randomly allocated to different studygroups. Randomization was performed based on a “matched distribution”method using the multi-task method (StudyDirector™ software, version3.1.399.19) randomized block design.

The fusion proteins were diluted according to the indicated dose and 10μl/kg of fusion proteins was injected intravenously to the mice onceevery week for three weeks. The mice were weighed twice per week afterrandomization. Tumor volumes were measured twice per week in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: “V=(L×W×W)/2, where V denoted tumor volume, L denoted tumorlength (the longest tumor dimension) and W denoted tumor width (thelongest tumor dimension perpendicular to L). Dosing as well as tumor andbody weight measurements were conducted in a Laminar Flow Cabinet. Thebody weights and tumor volumes were measured by using StudyDirectormsoftware (version 3.1.399.19).

To determine antitumor activity of the fusion proteins, tumor growthinhibition (TGI) percentages were measured and recorded daily using thefollowing formula TGI (%)=100×(1−T/C), where T and C denoted mean tumorvolume (or weight) of the treated and control groups respectively foreach of the fusion proteins and controls.

Of the fusion proteins tested, 1N-m3A7/IL-15 showed the most inhibitoryeffect on tumor growth (Table 23 and FIG. 24 ).

Taken together, the data obtained from Examples 25 and 26 suggests thatcompared to the C-terminal fusion antibodies, the N-terminal fusionantibodies reduce the toxicity further because of the lower stimulationfor the peripheral T cells. The data further shows that the N-terminalfusion molecules are particularly effective in stimulating tumorinfiltrating lymphocytes (TIL) as compared on peripheral T cells due tothe cis-presentation; the reduction of IL-2Rβγ binding in N-terminalfusion molecules allows the PD-1/IL-15 to selectively bind to TIL,therefore reducing its toxicity. Finally, the data shows that 1N-fusionmolecules are superior to the 2N-fusion molecules.

TABLE 23 Ability of anti-PD-L1-SD15 fusion proteins to prolong survivalof mice bearing PD-1 expressing tumors in PD-1/PD-L1 resistant LewisLung Model. RMP1-14 is a control, monoclonal antibody reacts with mousePD-1. Name 2 × IL15 1 × IL15 Antibody fusions fusioons RMP1-14 m3A7 2C2N 1C 1N Dose mg/kg 10 10 3.3 3.3 5.9 5.9 nmol/kg 66.7 66.7 17.5 17.535.0 35.0 Frequency, BIW, IP QW, IP QW, IP route Tumor growth −4.9 ± 9.6± 56.4 ± 49.7 ± 53.1 ± 75.4 ± inhibition, % 36.6 34.9 25.2 21.4 16.712.5

Example 27: hPD-1/IL-15 Fusion Proteins Retained their PD-1/PD-L1Inhibitory Function

Promega Blockade Bioassay was performed as described in Example 5 usingfusion proteins 1C-38B2-hSD15, 2C-38B2-hSD15, 1N-38B2-hSD15,2N-38B2-hSD15, 1N-65D-hSD15-38B2, and 1N-65S-hSD15-38B2. All fusionmolecules retained PD-1/PD-L1 inhibitory functions as compared to 38B2(FIG. 26 ). 1N-fusions provided the most PD-1/PD-L1 inhibitoryfunctions.

Example 28: hPD-1/IL-15 Fusion Proteins Stimulate IL-15 Activity

ExaM07e Culture and Proliferation Assay: To determine the ability of thefusion proteins to stimulate IL-15 in vitro, the human acutemegakaryoblastic leukemia M07e cell line was cultured in INDM mediumsupplemented with 15% heat-inactivated FBS and 20% conditioned medium ofcell line 5637.

Serial dilutions of antibodies or fusion proteins were added to a96-well plate, and then cells were seeded at 2.5×10⁴ per well in INDMmedium supplemented with 10% heat-inactivated FBS. The cells wereincubated at 37° C. for 3-5 days and cell proliferation was detectedusing CellTiter-Glo® luminescent cell viability assay kit (Promega).

While all tested fusion protein constructs stimulated IL-15 activity, INfusions showed a decreased ability to stimulate as compared to 1C, 2C,or 2N constructs (FIG. 27 ).

Example 29: Efficacy Study of hPD-L1/SD15 Fusion Molecules in CT26Syngeneic Models

To determine if the orientation or the number of IL-15/IL-15Rα sushidomains have any effect on the ability of the fusion molecules toinhibit tumor growth, an in vivo efficacy study was performed in a CT26syngeneic model using 1N-SD15-D7A8, 2N-SD15-D7A8, 1C-SD15-D7A8, and2C-SD15-D7A8. CT26 syngeneic model mice were treated with the fusionproteins and observed for tumor development/reduction. CT26 is a murinecolon carcinoma line.

Tumor volumes were measured twice per week in two dimensions using acaliper, and the volume was expressed in mm³ using the formula:“V=(L×W×W)/2, where V denoted tumor volume, L denoted tumor length (thelongest tumor dimension) and W denoted tumor width (the longest tumordimension perpendicular to L). Dosing as well as tumor and body weightmeasurements were conducted in a Laminar Flow Cabinet. The body weightsand tumor volumes were measured by using StudyDirectorm software(version 3.1.399.19).

Results of the efficacy study with PD-L1/IL-15 molecules are shown inFIG. 28 .

Example 30: The Presence of IL-5 Amino Acid Substitutions in the1N-Fusion Proteins Did not Affect the Fusion Protein's PD-1/PD-L1Inhibitory Function

Promega Blockade Bioassays (FIG. 26C, FIG. 29E, and FIG. 29F) and ELISAcompetition assays (FIGS. 29B and 29C) showed that N65S or N65D aminoacid substitutions in 1N-fusion proteins comprising IL-15 did not affectthe ability of the fusion proteins to effectively disrupt PD-1/PD-L1 orPD-1/PD-L2 interactions.

Similarly, no significant differences were observed in the strongbinding of 1N-fusion proteins comprising wild-type IL-15 or IL-15 N65S,respectively, to soluble PD-1 or cells expressing PD-1 (FIGS. 29A and29D).

Example 31: 1N-Fusion Proteins Comprising an IL-15 Amino AcidSubstitution Showed Weaker IL-15 Simulation In Vitro

The ability of 1N-fusion proteins to stimulate IL-15 activity in humanand in mouse cells was examined. In the presence of N65S-1N-IL-15/38B2,human acute megakaryoblastic leukemia cells (M07e, FIGS. 30A, 30B, and30C), hPBMCs (FIG. 30D), IL-2/IL-15-dependent murine lymphocyte celllines (CTLL2, FIG. 31A), murine spleen cells (FIG. 31B) grew slower thanin the presence of wild type 1N-IL-15/38B2. Binding of N65S-1N-/38B2 tohIL-2Rβ transfected HEK293 diminished (FIG. 30E).

Further, both 1N-IL-15/38B2 and mut-1N-IL-15/38B2 induced a better doseresponse in proliferating CD8⁺ T cells than CD4⁺ T cells in both mousespleen (FIG. 31C) and hPBMCs (FIG. 31D).

Example 32: 1N-Fusion Proteins Comprising an IL-15 Amino AcidSubstitution Exhibited Strong Anti-Tumor Activity In Vivo in anAnti-PD-1 Antibody-Resistant Mouse Model

In vivo efficacy of 1N-fusion proteins comprising wild-type IL-15, IL-15N65S, or IL-15 N65D, respectively, was evaluated in hPD-1/hPD-L1transgenic BALB/c mice bearing Keytruda (an anti-PD-1 antibody)resistant hPD-L1-CT26 tumors. The fusion antibodies (once per week) andcontrols (twice per week) were injected (intraperitoneal) when the tumorsize reached approximately 100 mms.

Both 1N-38B2/IL-15 and N65S-1N-38B2/IL-15 exhibited strong antitumoractivity (FIG. 32A). Anti-tumor efficacy of N65S-1N-38B2/IL-15 was dosedependent (FIG. 32B). 12 mg/kg, QW×3 weeks of N65S-1N-38B2/IL-15demonstrated tumor regression (FIGS. 32B and 32C). No weight loss ormortality was observed with N65S-IN-38B2/IL-15 when dosed at 12 mg/kg,but significant weight loss and mortality were observed with1N-38B2/IL-15 when dosed at >1 mg/kg (FIG. 32E and FIG. 32E). Anon-targeting 1N-IL-15 fusion did not show any anti-tumor efficacy.Significant weight loss (FIG. 32D) and mortality were observed whendosed >1 mg/kg for the non-targeting 1N-IL-15 fusion (data not shown).

1N-fusion proteins comprising IL-15 N65S showed improved efficacy ascompared to 1N-fusion proteins comprising IL-15 N65D (FIGS. 321 and32K).

In sum, fusion proteins comprising mutated IL-15 loweredIL-15-associated potency and stimulation, resulting in lower toxicityand an increased therapeutic treatment window.

Example 33: 1N-Fusion Proteins Comprising an IL-15 Amino AcidSubstitution Exhibited Strong Anti-Tumor Activity In Vivo in Mice thatwere Rechangelled with Tumor Cells

In vivo efficacy of 1N-fusion proteins comprising IL-15 N65S or IL-15N65D, respectively, was evaluated in hPD-1/hPD-L1 transgenic mice.

0.5×10⁶ of hPDL1/CT26 was subcutaneously inoculated to the right lowerflank of the mice. When the tumor size reached to about 100 mm³, themice were intraperitoneally injected with 12 mg/kg of N65S-1N-38B2/IL-15or N65D-1N-38B2/IL-15 once every week and twice every week for threeweeks respectively. The tumors in 5 mice (of 6) completely disappearedin about 25 days for N65S-1N-38B2/IL-15 treatment and 40 days forN65D-1N-38B2/IL-15 treatment.

At day 88, 0.5×10⁶ of hPDL1/CT26 cells and 0.1×10⁶ of CT26 cells weresubcutaneously inoculated to the left lower flank and left upper flankrespectively. No tumor growth was observed for both cell lines up to day135 days, showing that the fusion protein protect mice againstrechallenges with tumor cells (FIG. 33 ).

Example 34: 1N-Fusion Proteins Comprising an IL-15 Amino AcidSubstitution Exhibited Improved Anti-Tumor Activity in hPD1/PDL1 MiceBearing hPDL1-CT26 Tumor as Compared to Combinations of Non-TargetedFusion Protein and an Anti-PD-1 Antibody

0.5×10⁶ of hPDL1/CT26 cells were subcutaneously inoculated to the rightlower flank of the mice. When the tumor size reached to about 100 mm³,the mice were intraperitoneally injected with 10 mg/kg of 38B2, 12 mg/kgof N65S-1N-38B2/IL-15, and combination of 10 mg/kg of 38B2 with 12 mg/kgof N65S-1N-DP47/IL-15 once every week for three weeks respectively. Forthe combination treatment, two test articles were injected sequentiallywith one hour.

The ability to suppress tumor growth was compared for the followingconstructs: (1) N65S-1N-38B2/IL-15 fusion protein; (2) a non-targetedfusion N65S-1N-DP47/IL-15; (3) a combination of anti-PD-1 antibody 38B2and the non-targeted fusion (i.e., N65S-1N-DP47/IL-15); and (4)anti-PD-1 antibody 38B2 only.

Tumors grew significantly slower in the N65S-1N-38B2/IL-15 group ascompared to the combination group (FIG. 34A). 83% (5/6) of the mice weretumor-free in the N65S-1N-38B2/IL-15 fusion group, while no tumor-freemice were observed in the combination group and one tumor-free mouse wasobserved in the anti-PD-1 antibody 38B2 only group. (FIG. 34B). No bodyweight loss was observed for 65S-1N-IL15/38B2 fusion group, about −5%bodyweight loss for non-targeted fusion 65S-1N-IL15/DP47 group (FIG.34C).

This data illustrates the robust anti-tumor activity ofN65S-1N-38B2/IL-15 fusion proteins and highlights its bi-functionalityand its ability to bind to both PD1 and IL2Rβγ, which are co-expressedon TILs.

Example 35: Additional Administration of an Anti-PD-1 Antibody does notLead to a Further Increase in Anti-Tumor Activity of 1N-Fusion Proteins

0.5×10⁶ of hPDL1/CT26 cells were subcutaneously inoculated to the rightlower flank of the mice. When the tumor size reached to about 100 mm³,the mice were intraperitoneally injected with 10 mg/kg of 38B2, 12 mg/kgof N65S-1N-38B2/IL-15, and combination of 10 mg/kg of 38B2 with 12 mg/kgof N65S-1N-38B2/IL-15 once every week for three weeks respectively. Forthe combination treatment, two test articles were mixed first, theninjected to the mice.

The ability to suppress tumor growth was compared for the followingconstructs: (1) N65S-1N-38B2/IL-15 fusion protein; (2) a combination ofanti-PD-1 antibody 38B2 and N65S-1N-38B2/IL-15 fusion protein; and (3)anti-PD-1 antibody 38B2 only.

Tumor grew significantly more slowly in the N65S-1N-38B2/IL-15 group ascompared to the combination group (FIG. 35A). 83% (5/6) mice weretumor-free in the N65S-1N-38B2/IL-15 group, no tumor-free mice wereobserved in the combination group and one tumor-free mouse was observedin the antibody only (FIG. 35B). The bodyweight is shown in FIG. 35C.

This data shows that 1N-fusion proteins are more effective in reducingtumor growth than combinations of 1N-fusion proteins and anti-PD-1antibodies.

Example 36: N65S-1N-38B2/IL-15 Fusion Protein Showed a Long SerumHalf-Life in hPD1/PDL1 Transgenic Mice Bearing hPDL1-CT 26 Tumors

hPD1/PDL1 transgenic Balb/c mice were inoculated subcutaneously at theright lower flank with hPDL1/CT26 tumor cells (0.5×10⁶) in 0.1 mL ofPBS. The animals were randomized and treatments were started when theaverage tumor volume reaches approximately 83 mms. The 5.1 mg/kg (0.35μmol/kg) of 38B2, or 1 or 6 mg/kg (0.06 or 35 μmol/kg) ofN65S-1N-38B2/IL-15 were administrated to the mice eitherintraperitoneally (IP) or intravenously (IV). After dosing, blood wascollected at 0.2 h, 5 h, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, 120 h and 168h post injection and the purified serum were used to measure theantibody concentration by the traditional ELISA, in which human PD1 wasused as the captured protein and biotin-IL15 and anti-human Fab was usedseparately to detect the KD050 and 38B2 binding.

N65S-1N-38B2/IL-15 exhibited a similar serum half-life profile asantibody 38B2 in both IV (FIG. 36A) and IP (FIG. 36B) injection withdose response up to 120 h after treatment.

Example 37: Mechanism of Action Study in hPDL1/PD1 Transgenic BALB/cMice Bearing hPDL1-CT26 Tumors

0.5×10⁶ of hPDL1/CT26 cells were subcutaneously inoculated to the rightlower flank of the hPD1/PDL1 transgenic BALB/c mice. When the tumor sizereached to about 17 5 mms, the mice were intravenously injected with0.06 or 0.35 μmol/kg each of (1) N65S-1N-38B2/IL-1, (2)N65S-1N-DP47/IL-15 (non-targeted control), (3) antibody 38B2 and (4) thecombination of antibody 38B2 and N65S-1N-38B2/IL-15. The tumor size wasmeasured twice per week and mice were terminated at day 7 after dosing(FIG. 37A). Blood and draining lymph node (DLN) from all groups werecollected to perform flow cytometry. Tumors were collected to performTILs analysis (FIG. 37B). One-way ANOVA was used for statisticalanalysis. When a significant F-statistics (a ratio of treatment varianceto the error variance) was obtained, comparisons between differentgroups were carried out with Games-Howell (equal variances not assumed)or Tukey (equal variances assumed) test. Comparisons between vehicle andother groups, * p<0.05; ** p<0.01; *** p<0.001. Error bars representedStandard Error of Mean (SEM).

It was shown that N65S-1N-38B2/IL-15 bound to PD1 and IL2Rβγ expressingTIL and differently proliferated CD8+ T cells. Specifically, whenanalyzing the CD8/CD4 ratio in tumors, the blood, and draining lymphnodes (treated with antibody (1) 38B2, (2) PD1 targeted fusion proteinN65S-1N-38B2/IL-15, (3) non-targeted fusion protein N65S-1N-DP47/IL-15,or (4) a combination of 38B2 and N65S-1N-38B2/IL-15), it was found thatN65S-1N-38B2/IL-15 promoted CD8⁺ T cells growth in tumor, but not in theblood and draining lymph nodes. The CD8/CD4 ratio did not change asresult of the other treatments ( )1) 38B2, (2) non-targeted fusionprotein N65S-1N-DP47/IL-15, or (3) a combination of 38B2 andN65S-1N-38B2/IL-15)) (FIG. 37C).

Next, CD8 and CD4 subtypes in tumors (treated with antibody (1) 38B2,(2) PD1 targeted fusion protein N65S-1N-38B2/IL-15, (3) non-targetedfusion protein N65S-1N-DP47/IL-15, or (4) a combination of 38B2 andN65S-1N-38B2/IL-15) were analyzed. It was found that effector memory CD8T cells were significantly increased in the tumor for N65S-1N-38B2/IL-15reated samples and slightly increased for the 38B2 treated samples (FIG.37D). No difference was observed among the other T cell subtypes indifferent treatment groups.

Example 38: N65S-1N-m3A7/IL-15 (Surrogate) Efficacy Studies in MultipleMouse Syngeneic Tumor Models

The indicated amount of tumor cells (Table 24) in 0.1 mL of PBS wassubcutaneously inoculated to the right flank of the mice. The animalswere randomized and treatments were started when the average tumorvolume reaches approximately 100 mms (between 75-125 mm³). Tumor volumeswere measured 2 times per week in two dimensions using a caliper, andthe volume was expressed in mm³ using the formula: V=(L×W×W)/2, where Vis tumor volume, L is tumor length (the longest tumor dimension) and Wis tumor width (the longest tumor dimension perpendicular to L).

Significant anti-tumor efficacies were observed in all 12 models(TGI>50%) in single dose treatment (Table 24). Completed response (tumorfree) were found for CT26, EMT6, MC38, H22, A20 and Pan02 (Table 24).Data for EMT6 as shown as an illustrative example (FIG. 38 ).

TABLE 24 Results of N65S-IN-m3A7/IL-15 (surrogate) efficacy studies inmultiple mouse syngeneic tumor models. Number Cancer Mouse strain TGI(%) CR Model of cells type (sex) Dose, route (day) (%) p CT26 5 × 10⁵colorectal BALB/C (F)* 12 mg/kg, IP 93.0 (d17) 75.0 <0.0001 EMT6 5 × 10⁵breast BALB/C (F) 12 mg/kg, IP 79.7 (d21) 71.4 <0.0001 Renca 1 × 10⁶kidney BALB/C (F)  6 mg/kg, IP 52.4 (d18) — <0.0001 MC38 1 × 10⁶colorectal C57BL/6 (F)  6 mg/kg, IP 73.7 (d18) 12.5 <0.0001 Hepa1-6 5 ×10⁶ liver C57BL/6 (F)  6 mg/kg, IV 76.4 (d23) — <0.0001 H22 1 × 10⁶BALB/C (F)  6 mg/kg, IV 89.8 (d18) 30.0 <0.0001 LL2 3 × 10⁵ lung C57BL/6(F)  6 mg/kg, IP 68.3 (d15) — <0.0001 A20 5 × 10⁵ lymphoma BALB/C (F)  6mg/kg, IP 76.5 (d15) 40.0 <0.0001 B16- 2 × 10⁵ melanoma C57BL/6 (F)  6mg/kg, IV 46.7 (d13) — 0.0033 BL6 B16- 2 × 10⁵ C57BL/6 (F)  6 mg/kg, IV60.2 (d9) — 0.0003 F10 Pan02 3 × 10⁶ pancreatic C57BL/6 (F)  6 mg/kg, IV76.3 (d30) 30.0 0.0380 RM-1 1 × 10⁶ prostate C57BL/6 (M)  6 mg/kg, IV73.0 (d10) — <0.0001 F = female. M = male. TGI = tumor growthinhibition. CR = completed response.

TABLE 25 Sequences of the disclosure SEQ ID Identifier Sequence NOAntibodies isolated from phage display library 19B6 HCDR1 GYTFTGYYMH 119B6 HCDR2 IINPSGGSTSYAQKFQG 2 19B6 HCDR3 TAGYDWLPSGLGMDV 3 19B6 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ 4APGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKTAGYDWLPSGLGMDVWGQGTT VTVSS 19B10 HCDR1 GFTFSSYAMH 519B10 HCDR2 GISNSGGNTYYADSVKG 6 19B10 HCDR3 DNYYYYMDV 7 19B10 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQA 8PGKGLEWVSGISNSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNYYYYMDVWGKGTTVTVSS 23A8 HCDR1 GFTFSSSAMH 9 23A8 HCDR2GISGSGSSTYYADSVKG 10 23A8 HCDR3 DNNYYYYMDV 11 23 A8 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMHWVRQAP 12GKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDNNYYYYMDVWGKGTTVTVSS 23A11 HCDR1 GYTFTDYSLH 1323A11 HCDR2 EVIPMEDTAYYAQTFQG 14 23 A11 HCDR3 EPFTMVRGGRYYYYGMDV 1523A11 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYSLHWVRQA 16PGQGLEWMGEVIPMFDTAYYAQTFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREPFTMVRGGRYYYYGMDVWGQ GTTVTVSS 23H9 HCDR1 GGTFSSSVIS17 23H9 HCDR2 GIIPIFGTANYAQKFQG 18 23H9 HCDR3 EGLGCSGGSCYSGYYYGMDV 1923H9 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSSVISWVRQAP 20GQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGLGCSGGSCYSGYYYGMDVWGQ GTTVTVSS 24H9 HCDR1 GFTFSSYWMS21 24H9 HCDR2 AISGSGGSTYYADSVKG 22 24H9 HCDR3 SPLQWVDV 23 24H9 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA 24PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS 19B6 LCDR1 RASQSINSWLA 2519B6 LCDR2 YASSLQS 26 19B6 LCDR3 QQGYSVPLS 27 19B6 VLDIQMTQSPSSLSASVGDRVTITCRASQSINSWLAWYQQKPG 28KAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQGYSVPLSFGQGTKLEIK19B10 LCDR1 RASQSVSTWLA 29 19B10 LCDR2 AASNLET 30 19B10 LCDR3 QQTYSTPYT31 19B10 VL DIQMTQSPSSLSASVGDRVTITCRASQSVSTWLAWYQQKPG 32KAPKLLIYAASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQTYSTPYTFGQGTKLEIK23A8 LCDR1 RASQSIGTWLA 33 23A8 LCDR2 AASTLRS 34 23A8 LCDR3 QQSYSTPYT 3523 A8 VL DIQMTQSPSSLSASVGDRVTITCRASQSIGTWLAWYQQKPG 36KAPKLLIYAASTLRSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSTPYTFGQGTKLEIK23A11 LCDR1 RASQNINKNLN 37 23 A11 LCDR2 DASNLQS 38 23A11 LCDR3 QHSET 3923A11 VL DIQMTQSPSSLSASVGDRVTITCRASQNINKNLNWYQQKPG 40KAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQHSETFGQGTKVEIK23H9 LCDR1 RSSQSLLHSNGYNYLD 41 23H9 LCDR2 LSSHRAS 42 23H9 LCDR3MQGAHWPYT 43 23H9 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYL 44QKPGQSPQLLIYLSSHRASGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCMQGAHWPYTFGQGTKVEIK24H9 LCDR1 RASQGISSWLA 45 24H9 LCDR2 SASSLQS 46 24H9 LCDR3 QQANSFPFT 4724H9 VL DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPG 48KAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQANSFPFTFGPGTKVDIK23H9 derivatives obtained by light pair shuffling using the VH chain of 23H9 31B1 LCDR1 RSSQSLLHSNGYNYLD 41 31B1 LCDR2 EASSLHS 49 31B1 LCDR3MQGTHWPYT 50 31B1 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYL 51QKPGQSPQLLIYEASSLHSGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCMQGTHWPYTFGQGTKVEIK33C4 LCDR1 RSSQSLLHSNGYNYLD 41 33C4 LCDR2 EASSLHT 52 33C4 LCDR3MQGSHWPYT 53 33C4 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYL 54QKPGQSPQLLIYEASSLHTGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCMQGSHWPYTFGQGTKLEIK33G8 LCDR1 RSSQSLLHSNGYNYLD 41 33G8 LCDR2 QASTLGS 55 33G8 LCDR3MQGTHWPYT 56 33G8 VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYL 57QKPGQSPQLLIYQASTLGSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQGTHWPYTFGQGTKLEIK34C1 LCDR1 RSSESLLYSNGNTYLD 58 34C1 LCDR2 EVSNRAS 59 34C1 LCDR3MQGTRWPYT 60 34C1 VL DIVMTQSPLSLPVTPGEPASISCRSSESLLYSNGNTYLDWYL 61QKPGQSPQLLIYEVSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQGTRWPYTFGQGTKVEIK23H9 RSSX₁SLLX₂SNGX₃X₄YLD 62 derivatives wherein X₁ is Q or E;motif LCDR1 wherein X₂ is H or Y; wherein X₃ is Y or N; andwherein X₄ is T or N 23H9 X₅X₆SX₇X₈X₉X₁₀ 63 derivativeswherein X₅ is L, Q or E; wherein X₆ is S, A, or V;wherein X₇ is H, N, T, or S; motif LCDR2 wherein X₈ is R or L;wherein X₉ is G, A, or H; and wherein X₁₀ is S or T 23H9 MQGX₁₁X₁₂WPYT64 derivatives wherein X₁₁ is A, T, or S; and motif LCDR3wherein X₁₂ is H or R 23H9 DIVMTQSPLSLPVTPGEPASISC RSSX₁SLLX₂SNGX₃X₄YLD65 derivatives WYLQKPGQSPQLLIY motif VLX₅X₆SX₇X₈X₉X₁₀GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YC MQGX₁₁X₁₂WPYTFGQGTKVEIK24H9 derivatives obtained by light pair shuffling using the VH chain of 24H932A11 LCDR1 RASESISIWLA 66 32A11 LCDR2 DASNLET 67 32A11 LCDR3 QQADSFPFT68 32A11 VL DIQMTQSPSSLSASVGDRVTITCRASESISIWLAWYQQKPGK 69APKLLIYDASNLETGVPSRFSGSGSGTNFTLTISSLQPEDFAT YYCQQADSFPFTFGPGTKVDIK32D11 LCDR1 RASQSINIWLA 70 32D11 LCDR2 DASNLET 67 32D11 LCDR3 QQGYSFPFT71 32D11 VL DIQMTQSPSSLSASVGDRVTITCRASQSINIWLAWYQQKPGK 72APKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQGYSFPFTFGPGTKVDIK32D2 LCDR1 RASQGISRWLA 73 32D2 LCDR2 EASTLQS 74 32D2 LCDR3 QQAYSFPFT 7532D2 VL DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPG 76KAPKLLIYEASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQAYSFPFTFGQGTKVDIK32G6 LCDR1 RASQSISSWLA 77 32G6 LCDR2 DASTLQS 78 32G6 LCDR3 QQANSFPFT 4732G6 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPG 79KAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK38A10 LCDR1 RASQSISTWLA 80 38A10 LCDR2 DASRLQN 81 38A10 LCDR3 QQGDSFPFT82 38A10 VL DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPG 83KAPKLLIYDASRLQNGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQGDSFPFTFGPGTKVDIK38A11 LCDR1 RASQSISSWLA 77 38A11 LCDR2 DASTLQS 78 38A11 LCDR3 QQGNSFPFT84 38A11 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPG 85KAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQGNSFPFTFGPGTKVDIK38A4 LCDR1 RASQSISSWLA 77 38A4 LCDR2 DASNLQT 86 38A4 LCDR3 QQANSFPFT 4738A4 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPG 87KAPKLLIYDASNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGQGTRLEIK38A6 LCDR1 RASQDISRWLG 88 38A6 LCDR2 AASTLQS 89 38A6 LCDR3 QQANSFPFT 4738A6 VL DIQMTQSPSSLSASVGDRVTITCRASQDISRWLGWYQQKPG 90KAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKLEIK38A8 LCDR1 RASESISIWLA 66 38A8 LCDR2 DASNLET 67 38A8 LCDR3 QQANSFPFT 4738A8 VL DIQMTQSPSSLSASVGDRVTITCRASESISIWLAWYQQKPGK 91APKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQANSFPFTFGPGTKVDIK38B1 LCDR1 RASQSISTWLA 80 38B1 LCDR2 EASKLER 92 38B1 LCDR3 QQAYSFPFT 7538B1 VL DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPG 93KAPKLLIYEASKLERGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQAYSFPFTFGPGTKVDIK38B10 LCDR1 RASQSISTWLA 80 38B10 LCDR2 DASTLQT 94 38B10 LCDR3 QQGYSFPFT71 38B10 VL DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPG 95KAPKLLISDASTLQTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQGYSFPFTFGPGTKVDIK38B2 LCDR1 RASESISSWLA 96 38B2 LCDR2 DASSLES 97 38B2 LCDR3 QQGDSFPFT 8238B2 VL DIQMTQSPSSLSASVGDRVTITCRASESISSWLAWYQQKPGK 98APKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQGDSFPFTFGQGTKLEIK38C11 LCDR1 RASQSISRWLA 99 38C11 LCDR2 DASTVQS 100 38C11 LCDR3 QQANSFPFT47 38C11 VL DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPG 101KAPKLLISDASTVQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK38C6 LCDR1 RAGRDINKWVA 102 38C6 LCDR2 DASSLQS 103 38C6 LCDR3 QQANSFPFA104 38C6 VL DIQMTQSPSSLSASVGDRVTITCRAGRDINKWVAWYQQKPG 105KAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFAFGQGTKVEIK38G11 LCDR1 RASENISRWLA 106 38G11 LCDR2 DASSLQS 103 38G11 LCDR3QQANSFPFT 47 38G11 VL DIQMTQSPSSLSASVGDRVTITCRASENISRWLAWYQQKPG 107KAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK38G9 LCDR1 RVSQSISNWLA 108 38G9 LCDR2 DASSLQS 103 38G9 LCDR3 QQANSFPFT47 38G9 VL DIQMTQSPSSLSASVGDRVTITCRVSQSISNWLAWYQQKPG 109KAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFT 38H3 LCDR1RASQGISTWLA 110 38H3 LCDR2 EASRLQS 111 38H3 LCDR3 QQAYSFPFT 75 38H3 VLDIQMTQSPSSLSASVGDRVTITCRASQGISTWLAWYQQKPG 112KAPKLLIYEASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQAYSFPFTFGPGTKVDIK39A3 LCDR1 RASQSISSWLA 77 39A3 LCDR2 DASSLQS 103 39A3 LCDR3 QQGDSFPLT113 39A3 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPG 114KAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQGDSFPLTFGQGTKVEIK39B11 LCDR1 RASQSISSWLA 77 39B11 LCDR2 EASRLQS 111 39B11 LCDR3 QQANSFPFT47 39B11 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPG 115KAPKLLIYEASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK39B3 LCDR1 RASQSIGPWLA 116 39B3 LCDR2 DASNLET 67 39B3 LCDR3 QQANSFPFT 4739B3 VL DIQMTQSPSSLSASVGDRVTITCRASQSIGPWLAWYQQKPG 117KAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK39B6 LCDR1 RASEDISHWLA 118 39B6 LCDR2 DAKDLHP 119 39B6 LCDR3 QQANSFPFT47 39B6 VL DIQMTQSPSSLSASVGDRVTITCRASEDISHWLAWYQQKPG 120KAPKLLIYDAKDLHPGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGQGTRLEIK39F11 LCDR1 RASQSISTWLA 80 39F11 LCDR2 DASTLQS 78 39F11 LCDR3 QQANSFPFT47 39F11 VL DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPG 121KAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK39G5 LCDR1 RASQNINVWLA 122 39G5 LCDR2 DASSLQS 103 39G5 LCDR3 QQANSFPFT47 39G5 VL DIQMTQSPSSLSASVGDRVTITCRASQNINVWLAWYQQKPG 123KAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGPGTKVDIK39G8 LCDR1 RASQGISPWLA 124 39G8 LCDR2 DASKLET 125 39G8 LCDR3 QQAYSFPFT75 39G8 VL DIQMTQSPSSLSASVGDRVTITCRASQGISPWLAWYQQKPG 126KAPKLLIYDASKLETGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQAYSFPFTFGGGTKVEIK39H11 LCDR1 RASQSIGIWVA 127 39H11 LCDR2 DASNLQS 38 39H11 LCDR3 QQADSFPFT68 39H11 VL DIQMTQSPSSLSASVGDRVTITCRASQSIGIWVAWYQQKPG 128KAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQADSFPFTFGQGTKVEIK39H2 LCDR1 RASQNIGPWLA 129 39H2 LCDR2 DASTLHS 130 39H2 LCDR3 QQANSFPFT47 39H2 VL DIQMTQSPSSLSASVGDRVTITCRASQNIGPWLAWYQQKPG 131KAPKLLIYDASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQANSFPFTFGQGTRLEIK39H7 LCDR1 RASQSISNWLA 132 39H7 LCDR2 DASNLQA 133 39H7 LCDR3 QQAYSFPFT75 39H7 VL DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPG 134KAPKLLIYDASNLQAGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQAYSFPFTFGPGTKVDIK 24H9RX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₈WX₁₉X₂₀ 135 derivatives wherein X₁₃ is A or V;motif LCR1 wherein X₁₄ is S or G; wherein X₁₅ is Q, E, or R;wherein X₁₆ is G, S, D or N; wherein X₁₇ is G, S, or N;wherein X₁₈ is S, I, R, T, K, P, N,H, or V; wherein X₁₉ is L or V; andwherein X₂₀ is G or A 24H9 X₂₁AX₂₂X₂₃X₂₄X₂₅X₂₆ 136 derivativeswherein X₂₁ is S, D, E, or A; motif LCR2 wherein X₂₂ is S, or K;wherein X₂₃ is S, N, T, R, or D; wherein X₂₄ is L or V;wherein X₂₅ is Q, E, or H; and wherein X₂₆ is S, N, A, R, P, or T 24H9QQX₂₇X₂₈SFPX₂₉X₃₀ 137 derivatives wherein X₂₇ is A, or G; motif LCR3wherein X₂₈ is N, D, or Y; wherein X₂₉ is F or L; andwherein X₃₀ is A, or T 24H9DIQMTQSPSSLSASVGDRVTITCRX₁₃X₁₄X₁₅X₁₆IX₁₇X₁₈WX₁₉X₂₀ 138 derivativesWYQQKPGKAPKLLIYX₂₁AX₂₂X₂₃X₂₄X₂₅X₂₆GVPSRFSGSGSG motif VKTDFTLTISSLQPEDFATYYCQQX₂₇X₂₈SFPX₂₉X₃₀FGPGTKVDI (see SEQ ID KNOS: 135-137 for X_(n))Mouse/human PD-1 crossreactive antibodiy isolated from phage displayR3A9 HCDR1 GHTFTSYYIH 139 R3A9 HCDR2 GIINPSGGSTSYAQKFQG 140 R3A9 HCDR3DRAYGGAGDY 141 R3A9 VH QVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQA 142PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSS R3A9 LCDR1 RASQSVGTWLA 143R3A9 LCDR2 SASTLQS 144 R3A9 LCDR3 QQSYSAPLT 145 R3A9 VLDIQMTQSPSSLSASVGDRVTITCRASQSVGTWLAWYQQKPG 146KAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSAPLTFGGGTKVEIKR3A9 derivatives obtained by light pair shuffling using the VH chain of R3A9m3A7 LCDR1 RASRSISNWLA 147 m3A7 LCDR2 AASSLQS 148 m3A7 LCDR3 QQSYSAPVT149 m3A7 VL DIQMTQSPSSLSASVGDRVTITCRASRSISNWLAWYQQKPG 150KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSAPVTFGQGTKVEIKm2C1 LCDR1 RASRSISSWLA 151 m2C1 LCDR2 AASSLQS 148 m2C1 LCDR3 QQSYSIPAT152 m2C1 VL DIQMTQSPSSLSASVGDRVTITCRASRSISSWLAWYQQKPGK 153APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQSYSIPATFGQGTKVEIKm2A1 LCDR1 RASRGISSWLA 154 m2A1 LCDR2 AASSLQS 148 m2A1 LCDR3 QQSYSPPWT155 m2A1 VL DIQMTQSPSSLSASVGDRVTITCRASRGISSWLAWYQQKPG 156KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSPPWTFGQGTKVEIKm4A5 LCDR1 RASQSIDTWLA 157 m4A5 LCDR2 AASTLES 158 m4A5 LCDR3 QQSYRAPLT159 m4A5 VL DIQMTQSPSSLSASVGDRVTITCRASQSIDTWLAWYQQKPG 160KAPKLLIYAASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYRAPLTFGQGTKVEIKm4D1 LCDR1 RASQSISAWLA 161 m4D1 LCDR2 KASSLES 162 m4D1 LCDR3 QQSYSPPAT163 m4D1 VL DIQMTQSPSSLSASVGDRVTITCRASQSISAWLAWYQQKPG 164KAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSPPATFGGGTKVEIKm3C6 LCDR1 RASQSIDTWLA 157 m3C6 LCDR2 EASSLQG 165 m3C6 LCDR3 QQSYSTWT166 m3C6 VL DIQMTQSPSSLSASVGDRVTITCRASQSIDTWLAWYQQKPG 167KAPKLLIYEASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSTWTFGQGTKVEIKHeavy and light chains of 38B2 and m3A7 38B2 full lengthEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA 168 HCPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL (comprisesQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSAST 24H9 VH (SEQKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ID NO: 24))ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) 38B2 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA 169 (T350V/PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL T366L/QMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSAST K392L/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG T394W)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN LALAPGHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP (comprisesKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH 24H9 VH (SEQNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS ID NO: 24))NKALGAPIEKTISKAKGQPREPQVY

LPPSREEMTKNQVSL

CLVKGFYPSDIAVEWESNGQPENNY

T

PPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)38B2 full length DIQMTQSPSSLSASVGDRVTITCRASESISSWLAWYQQKPGK 170 LCAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFAT (comprisesYYCQQGDSFPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS 38B2 VL (SEQGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD ID NO: 98))SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC m3A7 fullQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQA 171 length mHCPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYM LALAPGELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSSAK (comprisesTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS R3A9 VHGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA (SEQ IDHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP NO: 142))KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) m3A7 mHCQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQA 172 (L351Y/PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYM F405A/ELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSSAK Y407V)TTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS LALAPGGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA (comprisesHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP R3A9 VHKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT (SEQ IDAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK NO: 142))DLGAPIERTISKPKGSVRAPQVYV

PPPEEEMTKKQVTLTCM VTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY

M

S KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) m3A7 fullDIQMTQSPSSLSASVGDRVTITCRASRSISNWLAWYQQKPG 173 length mLCKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA (comprisesTYYCQQSYSAPVTFGQGTKVEIKRADAAPTVSIFPPSSEQLT m3A7 VL (SEQSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ ID NO: 150))DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNECFusions of 38B2 heavy chains with SD15 hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 174 38B2 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG LALAPG GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI (comprisesDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD 24H9 VH (SEQTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS ID NO: 24), FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGG linker 1 (SEQGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV ID NO: 215),RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT and linker 2LYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS (SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO: 216))NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(K) hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 175 38B2HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGG LALAPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV (comprisesRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT 24H9 VH (SEQLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS ID NO: 24),ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW linker 1 (SEQNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC ID NO: 215),NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF and linker 2LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV (SEQ IDEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC NO: 216))KVSNKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS F

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(K) 38B2HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA 176 LALAPG +PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL hSD15QMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSAST (comprisesKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 24H9 VH (SEQALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN ID NO: 24) andHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP linker 1 (SEQKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH ID NO: 215))NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL TECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS GGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS 38B2 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA 177 (T350V/PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL L351Y/QMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSAST F405A/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Y407V)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN LALAPG +HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP hSD15KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH (comprisesNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS 24H9 VH (SEQNKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLT ID NO: 24) and CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

linker 1 (SEQ SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGS ID NO: 215))CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL TECVLNKATNVAHWTTPSLKC IRDPALVHQ RPAPPSGGS GGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS N65A hSD15+ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 178 38B2 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVE A LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGGGGGGGGGGSGGGGGGGGGGGG LALAPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV (comprisesRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT 24H9 VH (SEQLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS ID NO: 24) andASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW linker 1 (SEQNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC ID NO: 215),NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF linker 1 (SEQLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV ID NO: 215),EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC and linker 2KVSNKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQ (SEQ ID VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS NO: 216))F

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(K) N65D hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 179 38B2 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVE D LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFI N TSGGGGSGGGGSGGGGSGGGGSGGGGSGG LALAPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV (comprisesRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT 24H9 VH (SEQLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS ID NO: 24),ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW linker 1 (SEQNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC ID NO: 215),NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF and linker 2LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV (SEQ IDEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC N: 216))KVSNKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS F

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(K) N65S hSD15+ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 180 38B2 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVE S LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGGGGGGGGGGSGGGGGGGGGGGG LALAPG GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQA (comprisesPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN 24H9 VH (SEQSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSS ID NO: 24),ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW linker 1 (SEQNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC ID NO: 215),NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF and linker 2LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV (SEQ IDEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC N: 216))KVSNKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQ VSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGS F

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG(K)Fusions of m3A7 heavy chains with SD15 mSD15 +ITCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTST 181 m3A7 mHcLIECVINKNTNVAHWTTPSLKC IRDPALVHQRPAPPSGGS LALAPG GGGGSGGGSGGGGSLQNWIDVRYDLEKIESLIQSIHIDTT (comprisesLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETV R3A9 VHRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSF (SEQ ID IRIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG NO: 142), linkerSQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQ 1 (SEQ IDAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVY NO: 215), andMELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSSA linker 2 (SEQKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS ID N: 216))GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) mSD15 +ITCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTST 182 m3A7 mHcLIECVINKNTNVAHWTTPSLKC IRDPALVHQRPAPPSGGS (T366L/ GGGGSGGGSGGGGS LQNWIDVRYDLEKIESLIQSIHIDTT K392L/ LYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVT394W) RNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSF LALAPG IRIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG (comprisesSQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQ R3A9 VHAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVY (SEQ IDMELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSS NO: 142), linkerAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW 1 (SEQ IDNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN NO: 215), andVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIF linker 2 (SEQPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVH ID N: 216))TAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTL

C MVTDFMPEDIYVEWTNNGKTELNY

N

EPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG (K) m3A7 mHcQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQA 183 LALPG +PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYM mSD15ELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSSAK (comprisesTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS R3A9 VHGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA (SEQ IDHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP NO: 142) andKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT linker 1 (SEQAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK ID NO: 215))DLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGSCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLI ECVINKNTNVAHWTTPSLKCIRDPALVHQRPAPPSGGSG GGGSGGGSGGGGSLQ NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFI RIVQMFINTS m3A7 mHCQVQLVQSGAEVKKPGASVKVSCKASGHTFTSYYIHWVRQA 184 (T366L/PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYM K392L/ELSSLRSEDTAVYYCARDRAYGGAGDYWGQGTLVTVSSAK T394W) +TTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS mSD15GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVA LALAPGHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPP (comprisesKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHT R3A9 VHAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNK (SEQ IDDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTL

CM NO: 142) and VTDFMPEDIYVEWTNNGKTELNY

N

EPVLDSDGSYFMYS linker 1 (SEQ KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGSCID NO: 215)) PPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKC IRDPALVHQRPAPPSGGSG GGGSGGGSGGGGSLQNWIDVRYDLEKIESLIQSIHIDTTL YTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFI RIVQMFINTSHeavy and light chains of control antibody DP47 and fusions of DP47 heavy chains withSD15 DP47 mHC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 185 (L351Y/GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ F405A/MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAKTTAPS Y407V)VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS LALAPGGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS Residues 1-115:TKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV DP47 VHLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP IERTISKPKGSVRAPQVYV

PPPEEEMTKKQVTLLCMVTDF MPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY

M

SKLRV EKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) DP47 mLCEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG 186 Residues 1-108:QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA DP47 VLVYYCQQYGSSPLTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC mSD15 +ITCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTS 187 DP47 mHCTLIECVINKNTNVAHWTTPSLKC IRDPALVHQRPAPPSG LALAPG GSGGGGSGGGSGGGGSLQNWIDVRYDLEKIESLIQSIHID (comprisesTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNE DP47 VH (seeTVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFL SEQ ID QSFIRIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSG NO: 185), linkerGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV 1 (SEQ IDRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT NO: 215), andLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAK linker 2 (SEQTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS ID N: 216))GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) mSD15 +ITCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTST 188 DP47 mHCLIECVINKNTNVAHWTTPSLKC IRDPALVHQRPAPPSGGS (T366L/ GGGGSGGGSGGGGSLQNWIDVRYDLEKIESLIQSIHIDTT K392L/ LYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVT394W) RNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSF LALAPG IRIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG (comprisesSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA DP47 VH (seePGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL SEQ IDQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAKTTA NO: 185), linkerPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL 1 (SEQ IDSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPA NO: 215), andSSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKD linker 2 (SEQVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQ ID N: 216))THREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGA PIERTISKPKGSVRAPQVYV

PPPEEEMTKKQVTLLCMVTDF MPEDIYVEWTNNGKTELNY

N

EPVLDSDGSYFMYSKLRV EKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(K) DP47 mHcEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 189 LALAPG +GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ mSD15MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAKTTAPS (comprisesVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS DP47 VH (seeGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS SEQ IDTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV NO: 185) andLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT linker 1 (SEQHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP ID NO: 215))IERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGSCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVIN KNTNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGS GGGSGGGGSLQ NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQ MFINTS DP47 mHCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 190 (T366L/GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ K392L/MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAKTTAPS T394W)VYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS LALAPG +GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS mSD15TKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDV (comprisesLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQT DP47 VH (seeHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAP SEQ IDIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTL

CMVTDF NO: 185) and MPEDIYVEWTNNGKTELNY

N

EPVLDSDGSYFMYSKLRV linker 1 (SEQEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGSCPPPVS ID NO: 215))IEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVIN KNTNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSG GGSGGGGSLQ NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFI NTS DP47 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 191 (T350V/GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ T366L/MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPS K392L/VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS T394W)GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS LALAPGNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK (comprisesDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK DP47 VH (seeTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL SEQ ID GAPIEKTISKAKGQPREPQVY

LPPSREEMTKNQVSL

CLVK NO: 185)) GFYPSDIAVEWESNGQPENNY

T

PPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) DP47 LCEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG 192 Residues 1-108:QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA DP47 VLVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 193 DP47 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG LALPG GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI (comprisesDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD DP47 VH (seeTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS SEQ ID FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGG NO: 185), linkerGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR 1 (SEQ IDQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL NO: 215), andYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAST linker 2 (SEQKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ID N: 216))ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 194 DP47 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGGGGGGGGGGSGGGGGGGGGGGG LALAPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR (comprisesQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL DP47 VH (seeYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAST SEQ IDKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG NO: 185), linkerALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 1 (SEQ IDHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP NO: 215), andKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH linker 2 (SEQNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS ID N: 216))NKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) DP47 HC +EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 195 LALAPGGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ hSD15MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPS (comprisesVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS DP47 VH (seeGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS SEQ IDNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK NO: 185) andDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK linker 1 (SEQTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL ID NO: 215))GAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVL NKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGG SGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS DP47 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP 196 (T350V/GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ L351Y/MNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPS F405A/VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS Y407V)GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS LALAPG +NTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK hSD15DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK (comprisesTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL DP47 VH (seeGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLTCLVK SEQ ID GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLT NO: 185) and VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPP linker 1 (SEQMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC ID NO: 215) VLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGG GGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTS N65A-hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 197 DP47 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVE A LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGG LALAPG GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA (comprisesPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMN DP47 VH (seeSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPSVFPLA SEQ IDPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF NO: 185), linkerPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD 1 (SEQ IDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS NO: 215), andRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE linker 2 (SEQQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK ID N: 216)) TISKAKGQPREPQVY

PPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) N65D-hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 198 DP47 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/Y407V) TVE D LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS LALAPGFVHIVQMFINTS GGGGSGGGGSGGGGSGGGGSGGGGSGG (comprisesGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR DP47 VH (seeQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL SEQ IDYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAST NO: 185), linkerKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 1 (SEQ IDALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN NO: 215), andHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP linker 2 (SEQKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH ID N: 216))NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) N65S-hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 199 DP47 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVE S LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGG LALAPGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR (comprisesQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL DP47 VH (seeYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAST SEQ IDKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG NO: 185), linkerALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 1 (SEQ IDHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP NO: 215) andKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH linker 2 (SEQNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS ID N: 216))NKALGAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)Heavy and light chains of anti-PD-L1 antibody D7A8 and fusions of D7A8 heavy chainswith SD15 D7A8 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 200PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSS D7A8 fullEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 201 length HCPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ (comprisesMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST D7A8 VHKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (SEQ IDALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN NO: 200))HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) D7A8 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 202 (S354C/PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ T366W)MNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST LALAKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (comprisesALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN D7A8 VHHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP (SEQ IDKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NO: 200))NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPP

REEMTKNQVSL

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (K) D7A8 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 203 (T350V/PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ T366L/MNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST K392L/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG T394W)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN LALAHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP (comprisesKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH D7A8 VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS SEQ ID NKALPAPIEKTISKAKGQPREPQVY

LPPSREEMTKNQVSL

NO: 200)) CLVKGFYPSDIAVEWESNGQPENNY

T

PPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) D7A8 VLQSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHP 204GKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAED EADYYCSSYTSSSTRVFGTGTKVTVLGQPD7A8 full QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHP 205 length LCGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAED (comprisesEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSS D7A8 VL (SEQEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETT ID NO: 204))KPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTEC(S) hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 206 D7A8 HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG LALA GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI (comprisesDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD D7A8 VHTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS (SEQ ID FVHIVQMFINTSGGGGGGGGGGGGSGGGGSEVQLLESG NO: 200), linkerGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWV 1 (SEQ IDSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAED NO: 215), andTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPL the linker ofAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT SEQ ID N: 244)FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 207 D7A8HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (Y349C/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI T366S/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDL368A/ TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSEVQLLESG LALAGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWV (comprisesSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAED D7A8 VHTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPL (SEQ IDAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT NO: 200), linkerFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV 1 (SEQ IDDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI NO: 215), andSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE the linker ofEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE SEQ ID KTISKAKGQPREPQV

TLPPSREEMTKNQVSL

C

VKGFYPS N: 244)) DIAVEWESNGQPENNYKTTPPVLDSDGSFFL

SKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) hSD15 +ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS 208 D7A8HCSLTECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSG (T350V/ GSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHI L351Y/ DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDF405A/ TVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS Y407V) FVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSEVQLLESG LALAGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWV (comprisesSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAED D7A8 VHTAIYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPL (SEQ IDAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT NO: 200), linkerFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV 1 (SEQ IDDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI NO: 215), andSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE the linker ofEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE SEQ ID KTISKAKGQPREPQVY

PPSREEMTKNQVSLTCLVKGFYP N: 244)) SDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

SKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K) D7A8 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 209 LALA +PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ hSD15MNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST (comprisesKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG D7A8 VHALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN (SEQ IDHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP NO: 200) andKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH linker 1 (SEQNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS ID NO: 215))NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL TECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGS GGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS D7A8 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 210 (Y349C/PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ T366S/MNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST L368A/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Y407V)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN LALA+HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP hSD15KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH (comprisesNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS D7A8 VH NKALPAPIEKTISKAKGQPREPQV

TLPPSREEMTKNQVSL

(SEQ ID C

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

NO: 200) and SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC linker 1 (SEQPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ID NO: 215))ECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSGGSG GGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDAT LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS D7A8 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQA 211 (T350V/PGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQ L351Y/MNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSAST F405A/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG Y407V)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN LALA +HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP hSD15KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH (comprisesNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS D7A8 VHNKALPAPIEKTISKAKGQPREPQVY

PPSREEMTKNQVSLT (SEQ ID CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

L

NO: 200) and SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGC linker 1 (SEQPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ID NO: 215))ECVLNKATNVAHWTTPSLKC IRDPALVHQRPAPPSGGSG GGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDAT LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTSIL-15 and IL-15 Rα sequences Human IL-15NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK 212 (mature protein,CFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG residue N65 isCKECEELEEKNIKEFLQSFVHIVQMFINTS in bold) Human IL-15MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHA 213 Rα subunitDIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNV (signal peptideAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPS is underlined,GKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSH residue N60 isESSHGTPSQTTAKNWELTASASHQPPGVYPQHSDTTVAISTS in bold)TVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTW GTSSRDEDLENCSHHL IL-15Rα sushiCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTE 214 domainCVLNKATNVAHWTTPSLKC (residue corresponding to residue N60 in SEQ IDNO: 213 is shown in bold) Linker sequences Linker 1IRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQ 215 Linker 2GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 216 ExemplaryIRDPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG 217 linker ExemplaryIRDPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 218 linker ExemplaryGGGGSGGGGSGGGGSGGGGS 244 linker VH: heavy variable chain HC: (fulllength) heavy chain VL: light variable chain LC: (full length) lightchain mHC: murine (full length) heavy chain mLC: murine (full length)light chain SD15: bold LALA/LALAPG substitution: underlined (not bold)Substitution to stabilize heterodimer of two heavy chains: bold,underlight, italics Linkers: underlined Amino acid substitutions inIL-15: double underlined (K): optional lysine reside

TABLE 26 Sequences of the disclosure Dimer Light Fc amino acidsubstitution Heavy Heavy chain Name of HC chain type for heterodimerchain 1 2 (with IL-15) 2N_D7A8_hIL-15 homo 205 none none 2061N_D7A8_hIL-15 hetero 205 Knobs-into-holes 202 207 (CW-CSAV) ZW1(VYAV-VLLW) 203 208 2C_D7A8_hIL-15 homo 205 none none 209 1C_D7A8_hIL-15hetero 205 Knobs-into-holes 202 210 (CW-CSAV) ZW1 (VYAV-VLLW) 203 2112N_DP47_mIL-15 homo 186 none none 187 1N_DP47_mIL-15 hetero 186 ModifiedZW1 185 188 (VYAV-VLLW) 2C_DP47_mIL-15 homo 186 none none 1891C_DP47_mIL-15 hetero 186 Modified ZW1 185 190 (VYAV-VLLW)2N_m3A7_mIL-15 homo 173 none none 181 1N_m3A7_mIL-15 hetero 173 ModifiedZW1 172 182 (VYAV-VLLW) 2C_m3A7_mIL-15 homo 173 none none 1831C_m3A7_mIL-15 hetero 173 Modified ZW1 172 184 (VYAV-VLLW)2N_38B2_hIL-15 homo 170 none none 174 1N_38B2_hIL-15 hetero 170 ZW1(VYAV-VLLW) 169 175 2C_38B2_hIL-15 homo 170 none none 176 1C_38B2_hIL-15hetero 170 ZW1 (VYAV-VLLW) 169 177 2N_DP47_hIL-15 homo 192 none none 1931N_DP47_hIL-15 hetero 192 ZW1 (VYAV-VLLW) 191 194 2C_DP47_hIL-15 homo192 none none 195 1C_DP47_hIL-15 hetero 192 ZW1 (VYAV-VLLW) 191 196N65A_1N_38B2_hIL-15 hetero 170 ZW1 (VYAV-VLLW) 169 178N65D_1N_38B2_hIL-15 hetero 170 ZW1 (VYAV-VLLW) 169 179N65S_1N_38B2_hIL-15 hetero 170 ZW1 (VYAV-VLLW) 169 180N65A_1N_DP47_IL-15 hetero 192 ZW1 (VYAV-VLLW) 191 197N65D_1N_DP47_hIL-15 hetero 192 ZW1 (VYAV-VLLW) 191 198N65S_1N_DP47_hIL-15 hetero 192 ZW1 (VYAV-VLLW) 191 199

TABLE 27Exemplary embodiments of fusion proteins comprising antibody 38B2. All fusion proteins in this tablecomprise the light chain of 38B2. The individual sequences referenced in this table are provided inTable 28. As an example, in 1N-38B2_1, the first heavy chain comprises heterodimerization amino acidsubstitutions T350V/T366L/K392L/T394W and the second heavy chain comprises heterodimerization aminoacid substitutions T350V/L351Y/F405A/Y407V. In 1N-38B2_2, the first heavy chain comprises hetero-dimerization amino acid substitutions T350V/L351Y/F405A/Y407V and the second heavy chain comprisesheterodimerization amino acid substitutions T350V/T366L/K392L/T394W.Fusion Heavy chain 1 (HC1) Heavy chain 2 (HC2) Linker betweenFc engineering protein SEQ ID SEQ ID IL-15Rα sushi Effector name NO NameNO: Name and IL-15 region Heterodimerization 1N-38B2_1 220 38B2HC1-1 22465N-38B2HC2-1 SGGSGGGGSGGGSGGGGSLQ LALAPG ZW1 1N-38B2_2 221 38B2HC1-2225 65N-38B2HC2-2 SGGSGGGGSGGGSGGGGSLQ (VYAV-VLLW) 1N-38B2_3 22238B2HC1-3 226 65N-38B2HC2-3 SGGSGGGGSGGGSGGGGSLQ Knobs-into-holes1N-38B2_4 223 38B2HC1-4 227 65N-38B2HC2-4 SGGSGGGGSGGGSGGGGSLQ (CW-CSAV)65S-38B2_1 220 38B2HC1-1 228 65S-38B2HC2-1 SGGSGGGGSGGGSGGGGSLQ LALAPGZW1 65S-38B2_2 221 38B2HC1-2 229 65S-38B2HC2-2 SGGSGGGGSGGGSGGGGSLQ(VYAV-VLLW) 65S-38B2_3 222 38B2HC1-3 230 65S-38B2HC2-3SGGSGGGGSGGGSGGGGSLQ Knobs-into-holes 65S-38B2_4 223 38B2HC1-4 23165S-38B2HC2-4 SGGSGGGGSGGGSGGGGSLQ (CW-CSAV) 65S-38B2_5 220 38B2HC1-1236 65S-38B2HC2-5 SGGGGSGGGGSGGGSGGGGS LALAPG ZW1 65S-38B2_6 22138B2HC1-2 237 65S-38B2HC2-6 SGGGGSGGGGSGGGSGGGGS (VYAV-VLLW) 65S-38B2_7222 38B2HC1-3 238 65S-38B2HC2-7 SGGGGSGGGGSGGGSGGGGS Knobs-into-holes65S-38B2_8 223 38B2HC1-4 239 65S-38B2HC2-8 SGGGGSGGGGSGGGSGGGGS(CW-CSAV) 65D-38B2_1 220 38B2HC1-1 232 65D-38B2HC2-1SGGSGGGGSGGGSGGGGSLQ LALAPG ZW1 65D-38B2_2 221 38B2HC1-2 23365D-38B2HC2-2 SGGSGGGGSGGGSGGGGSLQ (VYAV-VLLW) 65D-38B2_3 222 38B2HC1-3234 65D-38B2HC2-3 SGGSGGGGSGGGSGGGGSLQ Knobs-into-holes 65D-38B2_4 22338B2HC1-4 235 65D-38B2HC2-4 SGGSGGGGSGGGSGGGGSLQ (CW-CSAV) 65S-38B2_9240 38B2HC1-3B 241 65S-38B2HC2-3B SGGSGGGGSGGGSGGGGSLQ LALAKnobs-into-holes (CW-CSAV) 65S-38B2_10 240 38B2HC1-3B 242 65S-38B2HC2-7BSGGGGSGGGGSGGGSGGGGS Knobs-into-holes (CW-CSAV) 65D-38B2_5 24038B2HC1-3B 243 65D-38B2HC2-3B SGGSGGGGSGGGSGGGGSLQ Knobs-into-holes(CW-CSAV)

TABLE 28 Sequences for fusion protein embodiments provided in Table 27.SEQ ID NO: Name Amino acid sequence 219 38B2 LCDIQMTQSPSSLSASVGDRVTITCRASESISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGS(comprisesGSGTDFTLTISSLQPEDFATYYCQQGDSFPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS38B2 VL (SEQ IDVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVNO: 98)) THQGLSSPVTKSFNRGEC 220 38B2 HC1-1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYAD (T350V/SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSV T366L/FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLK392L/ GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTC T394W)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS LALAPGNKAL

APIEKTISKAKGQPREPQVY V LPPSRDELTKNQVSL L CLVKGFYPSDIAVEWESNGQPENNY L T WPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 221 38B2 HC1-2EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYAD (T350V/SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSV L351Y/FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLF405A/ GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTC Y407V)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS LALAPGNKAL

APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 222 38B2 HC1-3EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYAD (CW-CSAV)SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSV S354C/FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLT366W GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTC LALAPGVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKAL

APIEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 223 38B2 HC1-4EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYAD (CW-CSAV)SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSV Y349C/FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLT366S/ GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTC L368A/VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS Y407VNKAL

APIEKTISKAKGQPREPQV C TLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYLALAPG KTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22465N-38B2 HC2-1ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL L351Y/ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG F405A/SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV Y407V)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 225 65N-38B2 HC2-2ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366L/ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG K392L/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV T394W)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY V LPPSRDELTKNQVS L L CLVKGFYPSDIAVEWESNGQPENNY L TW PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 22665N-38B2 HC2-3ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (CW-CSAV) RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAPGGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L S C AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 227 65N-38B2 HC2-4ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ S354C/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366WELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG LALAPG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYTLPP C RDELTKNQVS L WCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 228 65S-38B2 HC2-1ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL L351Y/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG F405A/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV Y407V)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 229 65S-38B2 HC2-2ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366L/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG K392L/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV T394W)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY V LPPSRDELTKNQVS L L CLVKGFYPSDIAVEWESNGQPENNY L TW PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 23065S-38B2 HC2-3ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (CW-CSAV) RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAPGGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L S C AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 231 65S-38B2 HC2-4ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ S354C/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366WELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG LALAPG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYTLPP C RDELTKNQVS L WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 232 65D-38B2 HC2-1ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL L351Y/ELQVISLESGDASIHDTVEDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG F405A/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV Y407V)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY VY PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF A L V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 233 65D-38B2 HC2-2ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (T350V/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366L/ELQVISLESGDASIHDTVEDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG K392L/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV T394W)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY V LPPSRDELTKNQVS L L CLVKGFYPSDIAVEWESNGQPENNY L TW PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 23465D-38B2 HC2-3ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (CW-CSAV) RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVEDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAPGGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L S C AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 235 65D-38B2 HC2-4ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ S354C/ RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366WELQVISLESGDASIHDTVEDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG LALAPG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYTLPP C RDELTKNQVS L WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 236 65S-38B2 HC2-5ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ(T350V/ RPAPP SGGGGSGGGGSGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL L351Y/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG F405A/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV Y407V)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY VY PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF A L V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 237 65S-38B2 HC2-6ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ(T350V/RPAPPSGGGGSGGGGSGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLT366L/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG K392L/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV T394W)RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP LALAPGLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVY V LPPSRDELTKNQVS L L CLVKGFYPSDIAVEWESNGQPENNY L TW PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 23865S-38B2 HC2-7ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ(CW-CSAV) RPAPP SGGGGSGGGGSGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAPGGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L S C AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 239 65S-38B2 HC2-8ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQS354C/ RPAPP SGGGGSGGGGSGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL T366WELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG LALAPG SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREPQVYTLPP C RDELTKNQVS L WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 240 38B2 HC1-3BEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSAISGSGGSTYYAD (CW-CSAV)SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSPLQWVDVWGQGTTVTVSSASTKGPSV S354C/FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLT366W GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTC LALAVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 24165S-38B2 HC2-3BITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (CW-CSAV) RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L SC A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 242 65S-38B2 HC2-7BITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ(CW-CSAV) RPAPP SGGGGSGGGGSGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVESLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L SC A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 243 65D-38B2 HC2-3BITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQ (CW-CSAV) RPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLL Y349C/ELQVISLESGDASIHDTVEDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGG T366S/ SGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV L368A/RQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSP Y407VLQWVDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LALAGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP E

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVS L SC A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variable chains are in bold (not underlined). LALAPGand LALA amino acid substitutions are indicated in bold and italics.Amino acid substitutions relating to heterodimerization are indicated inbold and underlined. The SD15 portion is shown in italics. Linkerregions are indicated in italics and underlined. All 38B2 HC comprise24H9 VH (SEQ ID NO: 24).

1-76. (canceled)
 77. An anti-PD-1 antibody, or an antigen-bindingfragment thereof, wherein: a. the sequences of CDR1H, CDR2H and CDR3Hcomprise SEQ ID NOS: 21, 22, and 23; and b. the sequences of CDR1L,CDR2L and CDR3L comprise: i. SEQ ID NOS: 96, 97 and 82 ii. SEQ ID NOS:45, 46, and 47; iii. SEQ ID NOS: 66, 67, and 68; iv. SEQ ID NOS: 70, 67,and 71; v. SEQ ID NOS: 73, 74, and 75; vi. SEQ ID NOS: 77, 78, and 47;vii. SEQ ID NOS: 80, 81, and 82; viii. SEQ ID NOS: 77, 78, and 84; ix.SEQ ID NOS: 77, 86, and 47; x. SEQ ID NOS: 88, 89, and 47; xi. SEQ IDNOS: 66, 67, and 47; xii. SEQ ID NOS: 80, 92, and 75; xiii. SEQ ID NOS:80, 94, and 71; xiv. SEQ ID NOS: 99, 100, and 47; xv. SEQ ID NOS: 102,103, and 104; xvi. SEQ ID NOS: 106, 103, and 47; xvii. SEQ ID NOS: 108,103, and 47; xviii. SEQ ID NOS: 110, 111, and 75; xix. SEQ ID NOS: 77,103, and 113; xx. SEQ ID NOS: 77, 111, and 47; xxi. SEQ ID NOS: 116, 67,and 47; xxii. SEQ ID NOS: 118, 119, and 47; xxiii. SEQ ID NOS: 80, 78,and 47; xxiv. SEQ ID NOS: 122, 103 and 47; xxv. SEQ ID NOS: 124, 125,and 75; xxvi. SEQ ID NOS: 127, 38, and 68; xxvii. SEQ ID NOS: 129, 130,and 47; or xxviii. SEQ ID NOS: 132, 133, and
 75. 78. The anti-PD-1antibody, or an antigen-binding fragment thereof, of claim 77, wherein:a. the heavy chain variable region comprises: i. SEQ ID NO: 24, or ii. asequence that is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO: 24; and b. thelight chain variable region comprises: i. SEQ ID NO: 48, 69, 72, 76, 79,83, 85, 87, 90, 91, 93, 95, 98, 101, 105, 107, 109, 112, 114, 115, 117,120, 121, 123, 126, 128, 131, or 134, or ii. a sequence that is at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to any of SEQ ID NO: 48, 69, 72, 76, 79, 83, 85, 87, 90,91, 93, 95, 98, 101, 105, 107, 109, 112, 114, 115, 117, 120, 121, 123,126, 128, 131, or
 134. 79. The anti-PD-1 antibody, or an antigen-bindingfragment thereof, of claim 77, wherein: the sequence of CDR1H comprisesthe sequence of SEQ ID NO: 21; the sequence of CDR2H comprises thesequence of SEQ ID NO: 22; the sequence of CDR3H comprises the sequenceof SEQ ID NO: 23 the sequence of CDR1L comprises the sequence of SEQ IDNO: 96; the sequence of CDR2L comprises the sequence of SEQ ID NO: 97;and the sequence of CDR3L comprises the sequence of SEQ ID NO:
 82. 80.The anti-PD-1 antibody, or an antigen-binding fragment thereof, of claim79, wherein: a. the heavy chain variable region comprises SEQ ID NO: 24,or a sequence that is at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 24; and b.the light chain variable region comprises SEQ ID NO: 98, or a sequencethat is at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to SEQ ID NO:
 98. 81. An anti-PD-1antibody, or an antigen-binding fragment thereof, wherein: a. thesequences of CDR1H, CDR2H and CDR3H comprise SEQ ID NOS: 17, 18, and 19;and b. the sequences of CDR1L, CDR2L and CDR3L comprise: i. SEQ ID NOS:41, 49, and 50; ii. SEQ ID NOS: 41, 42, and 43; iii. SEQ ID NOS: 41, 52,and 53; iv. SEQ ID NOS: 41, 55, and 56; or v. SEQ ID NOS: 58, 59, and60.
 82. The anti-PD-1 antibody, or an antigen-binding fragment thereof,of claim 81, wherein: a. the heavy chain variable region comprises: i.SEQ ID NO: 20, or ii. a sequence that is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 20; and b. the light chain variable region comprises: i. SEQ IDNO: 44, 51, 54, 57, or 61, or ii. a sequence that is at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to any one of SEQ ID NO: 44, 51, 54, 57, or
 61. 83. Theanti-PD-1 antibody, or an antigen-binding fragment thereof, of claim 77,wherein the antibody or antigen-binding fragment is a multispecific or abispecific antibody or antigen-binding fragment thereof.
 84. Theanti-PD-1 antibody, or an antigen-binding fragment thereof, of claim 77,wherein the antibody or antigen-binding fragment is an scFv, Fv, Fab′,Fab, F(ab′)2, or diabody.
 85. The anti-PD-1 antibody, or anantigen-binding fragment thereof, of claim 77, wherein the antibody isan IgG class immunoglobulin, optionally having isotype IgG1 or IgG4. 86.The anti-PD-1 antibody, or an antigen-binding fragment thereof, of claim77, wherein the antibody or antigen-binding fragment comprises a firstand a second heavy chain constant region, and wherein the antibody orantigen-binding fragment thereof comprises at least one modification inthe CH3 domains of the first and the second heavy chain causingheterodimerization.
 87. The anti-PD-1 antibody, or an antigen-bindingfragment thereof, of claim 86, wherein: a. the CH3 domain of the firstheavy chain comprises one or more of amino acid substitutions S354C andT366W; and the CH3 domain of the second heavy chain comprises one ormore of amino acid substitutions Y349C, T366S, L368A, and Y407V; b. theCH3 domain of the first heavy chain comprises one or more of amino acidsubstitutions T350V, L351Y, F405A, and Y407V; and the CH3 domain of thesecond heavy chain comprises one or more of amino acid substitutionsT350V, T366L, K392L, and T394W; or c. the CH3 domain of the first heavychain comprises one or more of amino acid substitutions L351Y, F405A,and Y407V; and the CH3 domain of the second heavy chain comprises one ormore of amino acid substitutions T366L, K392L, and T394W (Kabat EU indexnumbering).
 88. The anti-PD-1 antibody, or an antigen-binding fragmentthereof, of claim 86, wherein the Fc domain, if present, comprises oneor more amino acid substitution that reduces binding to an Fc receptoror reduces effector function, wherein the one or more amino acidsubstitution is selected from L234A, L235A, and P329G (Kabat EU indexnumbering).
 89. A fusion protein comprising: a. the anti-PD-1 antibody,or an antigen-binding fragment thereof, of claim 77; b. an IL-15Rα sushidomain polypeptide comprising the amino acid sequence of SEQ ID NO: 214,or an amino acid sequence that is at least 95% identical to SEQ ID NO:214; c. an IL-15 polypeptide comprising the amino acid sequence of SEQID NO: 212, or an amino acid sequence that is at least 90% or at least95% identical to SEQ ID NO: 212; and d. a first linker polypeptidejoining the IL-15Rα sushi domain polypeptide and the IL-15 polypeptide,optionally wherein the IL-15Rα sushi domain polypeptide is fused to theN-terminus of the IL-15 polypeptide.
 90. The fusion protein of claim 89,wherein (i) the IL-15Rα sushi domain polypeptide and the IL-15polypeptide are fused to (ii) the N-terminus of the heavy chain variableregion, optionally by a second linker.
 91. The fusion protein of claim90, wherein the first linker (i) is between 5 and 40 amino acids long,optionally, consists predominantly of Gly (G), Asn (N), Ser (S), Thr(T), Ala (A), Leu (L), and Gln (Q); and/or (ii) comprises a sequenceselected from the group consisting of SEQ ID NOS: 215-218 and
 244. 92.The fusion protein of claim 91, wherein the second linker (i) is between5 and 40 amino acids long, optionally, consists predominantly of Gly(G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L), and Gln (Q), and/or(ii) comprises a sequence selected from the group consisting of SEQ IDNOS: 215-218 and
 244. 93. The fusion protein of claim 92, wherein thefusion protein comprises not more than one IL-15Rα sushi domainpolypeptide and not more than one IL-15 polypeptide.
 94. The fusionprotein of claim 92, wherein the IL-15 polypeptide is a mutant IL-15polypeptide comprising one or more amino acid substitutions at positions45, 65, or 108 as numbered according to the mature human IL-15 sequenceof SEQ ID NO: 212, wherein the one or more amino acid substitutions inthe IL-15 polypeptide are selected from L45A, N65A, N65D, N65S, N65K,and Q108S as numbered according to the mature human IL-15 sequence ofSEQ ID NO:
 212. 95. A fusion protein comprising a light chain sequencecomprising SEQ ID NO: 219 or SEQ ID NO: 98 and a first heavy chainsequence comprising SEQ ID NO: 240 and a second heavy chain sequencecomprising SEQ ID NO:
 241. 96. A nucleic acid sequence encoding theanti-PD-1 antibody, or an antigen-binding fragment thereof of claim 77.97. A cell comprising the nucleic acid of claim
 96. 98. A vectorcomprising nucleic acids encoding the light and heavy chains encodingthe anti-PD-1 antibody, or an antigen-binding fragment thereof, of claim77.
 99. A pharmaceutical composition comprising (i) the anti-PD-1antibody, or an antigen-binding fragment thereof, of claim 77; and (ii)a pharmaceutically acceptable carrier.
 100. A method of inhibitingbinding of PD-1 to a ligand of PD-1, optionally PD-L1 or PD-L2, in asubject in need thereof, the method comprising administering to thesubject an effective amount of the anti-PD-1 antibody, or anantigen-binding fragment thereof, of claim
 77. 101. A method forincreasing T cell activation or stimulating the immune system in asubject in need thereof, the method comprising administering to thesubject an effective amount of the anti-PD-1 antibody, or anantigen-binding fragment thereof, of claim
 77. 102. A method of treatingcancer or reducing tumor growth or tumor metastasis in a subject in needthereof, the method comprising administering to the subject an effectiveamount of the anti-PD-1 antibody, or an antigen-binding fragmentthereof, of claim
 77. 103. A method of inhibiting binding of PD-1 to aligand of PD-1, optionally PD-L1 or PD-L2, in a subject in need thereof,the method comprising administering to the subject an effective amountof the fusion protein of claim
 89. 104. A method for increasing T cellactivation or stimulating the immune system in a subject in needthereof, the method comprising administering to the subject an effectiveamount of the fusion protein of claim
 89. 105. A method of treatingcancer or reducing tumor growth or tumor metastasis in a subject in needthereof, the method comprising administering to the subject an effectiveamount of fusion protein of claim 89.